Use of inhibitors of bruton&#39;s tyrosine kinase (BTK)

ABSTRACT

Disclosed herein are methods for treating a cancer comprising: a. administering a Btk inhibitor to a subject sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes defined by immunophenotyping; b. determining the expression profile of one or more biomarkers from one or more subpopulation of lymphocytes; and c. administering a second agent based on the determined expression profile.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/869,700, filed Apr. 24, 2013; which is a continuation of U.S.application Ser. No. 13/153,317, filed Jun. 3, 2011; which claims thebenefit of priority from U.S. Provisional Patent Application No.61/351,130, filed Jun. 3, 2010; U.S. Provisional Patent Application No.61/351,655, filed Jun. 4, 2010; U.S. Provisional Patent Application No.61/351,793, filed Jun. 4, 2010; U.S. Provisional Patent Application No.61/351,762, filed Jun. 4, 2010; U.S. Provisional Patent Application No.61/419,764, filed Dec. 3, 2010; and U.S. Provisional Patent ApplicationNo. 61/472,138, filed Apr. 5, 2011; all of which are herein incorporatedby reference in their entirety.

BACKGROUND OF THE INVENTION

Bruton's tyrosine kinase (Btk), a member of the Tec family ofnon-receptor tyrosine kinases, is a key signaling enzyme expressed inall hematopoietic cells types except T lymphocytes and natural killercells. Btk plays an essential role in the B-cell signaling pathwaylinking cell surface B-cell receptor (BCR) stimulation to downstreamintracellular responses.

Btk is a key regulator of B-cell development, activation, signaling, andsurvival (Kurosaki, Curr Op Imm, 2000, 276-281; Schaeffer andSchwartzberg, Curr Op Imm 2000, 282-288). In addition, Btk plays a rolein a number of other hematopoietic cell signaling pathways, e.g., Tolllike receptor (TLR) and cytokine receptor-mediated TNF-α production inmacrophages, IgE receptor (FcepsilonRI) signaling in Mast cells,inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells,and collagen-stimulated platelet aggregation. See, e.g., C. A. Jeffries,et al., (2003), Journal of Biological Chemistry 278:26258-26264; N. J.Horwood, et al., (2003), The Journal of Experimental Medicine197:1603-1611; Iwaki et al. (2005), Journal of Biological Chemistry280(48):40261-40270; Vassilev et al. (1999), Journal of BiologicalChemistry 274(3):1646-1656, and Quek et al. (1998), Current Biology8(20):1137-1140.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, the hematological malignancy is CLL. In someembodiments, the treating the hematological malignancy comprisesmanaging the hematological malignancy. In some embodiments, thehematological malignancy is a B-cell malignancy. In some embodiments,the hematological malignancy is a leukemia, lymphoproliferativedisorder, or myeloid. In some embodiments, the mobilized cells aremyeloid cells or lymphoid cells. In some embodiments, analyzing themobilized plurality of cells comprises measuring the peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the peripheral blood concentration of the mobilizedplurality of cells increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein peripheral blood concentration of the mobilized plurality of cells.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the peripheral bloodconcentration of the mobilized plurality of cells as compared to theconcentration before administration of the Btk inhibitor. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells has increased for a predetermined length oftime. In some embodiments, analyzing the mobilized plurality of cellscomprises counting the number of mobilized plurality of cells in theperipheral blood. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the number ofmobilized plurality of cells in the peripheral blood increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises preparing abiomarker profile for a population of cells isolated from the pluralityof cells, wherein the biomarker profile indicates the expression of abiomarker, the expression level of a biomarker, mutations in abiomarker, or the presence of a biomarker. In some embodiments, thebiomarker is any cytogenetic, cell surface molecular or protein or RNAexpression marker. In some embodiments, the biomarker is: ZAP70;t(14,18); β-2 microglobulin; p53 mutational status; ATM mutationalstatus; del(17)p; del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25;CD38; CD103; CD138; secreted, surface or cytoplasmic immunoglobulinexpression; V_(H) mutational status; or a combination thereof. In someembodiments, the method further comprises providing a second cancertreatment regimen based on the biomarker profile. In some embodiments,the method further comprises not administering based on the biomarkerprofile. In some embodiments, the method further comprises predictingthe efficacy of a treatment regimen based on the biomarker profile. Insome embodiments, the hematological malignancy is a chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, or anon-CLL/SLL lymphoma. In some embodiments, the hematological malignancyis follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma,marginal zone lymphoma, Burkitt's lymphoma, non-Burkitt high grade Bcell lymphoma, or extranodal marginal zone B cell lymphoma. In someembodiments, the hematological malignancy is chronic myelogenous (ormyeloid) leukemia, or acute lymphoblastic leukemia. In some embodiments,the hematological malignancy is relapsed or refractory diffuse largeB-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma,relapsed or refractory follicular lymphoma, relapsed or refractory CLL;relapsed or refractory SLL; relapsed or refractory multiple myeloma. Insome embodiments, the Btk inhibitor forms a covalent bond with acysteine sidechain of a Bruton's tyrosine kinase, a Bruton's tyrosinekinase homolog, or a Btk tyrosine kinase cysteine homolog. In someembodiments, the irreversible Btk inhibitor is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.In some embodiments, the amount of the irreversible Btk inhibitor isfrom 300 mg/day up to, and including, 1000 mg/day. In some embodiments,the amount of the irreversible Btk inhibitor is from 420 mg/day up to,and including, 840 mg/day. In some embodiments, the amount of theirreversible Btk inhibitor is about 420 mg/day, about 560 mg/day, orabout 840 mg/day. In some embodiments, the amount of the irreversibleBtk inhibitor is about 420 mg/day. In some embodiments, the AUC₀₋₂₄ ofthe Btk inhibitor is between about 150 and about 3500 ng*h/mL. In someembodiments, the AUC₀₋₂₄ of the Btk inhibitor is between about 500 andabout 1100 ng*h/mL. In some embodiments, the Btk inhibitor isadministered orally. In some embodiments, the Btk inhibitor isadministered once per day, twice per day, or three times per day. Insome embodiments, the Btk inhibitor is administered until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered daily until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered every other day untildisease progression, unacceptable toxicity, or individual choice. Insome embodiments, the Btk inhibitor is a front line therapy, second linetherapy, third line therapy, fourth line therapy, fifth line therapy, orsixth line therapy. In some embodiments, the Btk inhibitor treats arefractory hematological malignancy. In some embodiments, the Btkinhibitor is a maintenance therapy. In some embodiments, the secondcancer treatment regimen comprises a chemotherapeutic agent, a steroid,an immunotherapeutic agent, a targeted therapy, or a combinationthereof. In some embodiments, the second cancer treatment regimencomprises a B cell receptor pathway inhibitor. In some embodiments, theB cell receptor pathway inhibitor is a CD79A inhibitor, a CD79Binhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3Kinhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or acombination thereof. In some embodiments, the second cancer treatmentregimen comprises an antibody, B cell receptor signaling inhibitor, aPI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, aradioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, ahistone deacetylase inhibitor, a protein kinase inhibitor, a hedgehoginhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1/2inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or acombination thereof. In some embodiments, the second cancer treatmentregimen comprises chlorambucil, ifosphamide, doxorubicin, mesalazine,thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine,fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab,dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab,bortezomib, pentostatin, endostatin, or a combination thereof. In someembodiments, the second cancer treatment regimen comprisescyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, andoptionally, rituximab. In some embodiments, the second cancer treatmentregimen comprises bendamustine, and rituximab. In some embodiments, thesecond cancer treatment regimen comprises fludarabine, cyclophosphamide,and rituximab. In some embodiments, the second cancer treatment regimencomprises cyclophosphamide, vincristine, and prednisone, and optionally,rituximab. In some embodiments, the second cancer treatment regimencomprises etoposide, doxorubicin, vinristine, cyclophosphamide,prednisolone, and optionally, rituximab. In some embodiments, the secondcancer treatment regimen comprises dexamethasone and lenalidomide. Insome embodiments, the inhibitor of Bruton's tyrosine kinase is areversible inhibitor. In some embodiments, the inhibitor of Bruton'styrosine kinase is an irreversible inhibitor. In some embodiments, theinhibitor of Bruton's tyrosine kinase forms a covalent bond with acysteine sidechain of a Bruton's tyrosine kinase, a Bruton's tyrosinekinase homolog, or a Btk tyrosine kinase cysteine homolog. In someembodiments, the inhibitor of Bruton's tyrosine kinase has the structureof Formula (D):

wherein:L_(a) is CH₂, O, NH or S;Ar is a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl;Y is an optionally substituted group selected from among alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;Z is C(═O), OC(═O), NHC(═O), C(═S), S(═O)_(x), OS(═O)_(x), NHS(═O)_(x),where x is 1 or 2;R₇ and R₈ are independently H; orR₇ and R₈ taken together form a bond;R₆ is H; and pharmaceutically active metabolites, or pharmaceuticallyacceptable solvates, pharmaceutically acceptable salts, orpharmaceutically acceptable prodrugs thereof. In some embodiments, theBruton's tyrosine kinase inhibitor is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.In some embodiments, La is O. In some embodiments, Ar is phenyl. In someembodiments, Z is C(═O), NHC(═O), or S(═O)₂. In some embodiments, eachof R₇ and R₈ is H. In some embodiments, Y is a 4-, 5-, 6-, or 7-memberedcycloalkyl ring; or Y is a 4-, 5-, 6-, or 7-membered heterocycloalkylring.

Disclosed herein, in certain embodiments, is a method for treatingrelapsed or refractory non-Hodgkin's lymphoma in an individual in needthereof, comprising: administering to the individual atherapeutically-effective amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.In some embodiments, the non-Hodgkin's lymphoma is relapsed orrefractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractorymantle cell lymphoma, or relapsed or refractory follicular lymphoma. Insome embodiments, the amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis from 300 mg/day up to, and including, 1000 mg/day. In someembodiments, the amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis from 420 mg/day up to, and including, 840 mg/day. In someembodiments, the amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis about 420 mg/day, about 560 mg/day, or about 840 mg/day. In someembodiments, the amount of the irreversible Btk inhibitor is about 420mg/day. In some embodiments, the AUC₀₋₂₄ of the Btk inhibitor is betweenabout 150 and about 3500 ng*h/mL. In some embodiments, the AUC₀₋₂₄ ofthe Btk inhibitor is between about 500 and about 1100 ng*h/mL. In someembodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered orally. In some embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered once per day, twice per day, or three times per day. Insome embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered until disease progression, unacceptable toxicity, orindividual choice. In some embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered until disease progression, unacceptable toxicity, orindividual choice. In some embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered daily until disease progression, unacceptable toxicity,or individual choice. In some embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis administered every other day until disease progression, unacceptabletoxicity, or individual choice. In some embodiments,(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-oneis a second line therapy, third line therapy, fourth line therapy, fifthline therapy, or sixth line therapy. In some embodiments, the Btkinhibitor is a maintenance therapy. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen. Insome embodiments, the second cancer treatment regimen is administeredafter mobilization of a plurality of lymphoid cells from thenon-Hodgkin's lymphoma. In some embodiments, the second cancer treatmentregimen is administered after lymphocytosis of a plurality of lymphoidcells from the non-Hodgkin's lymphoma. In some embodiments, the secondcancer treatment regimen comprises a chemotherapeutic agent, a steroid,an immunotherapeutic agent, a targeted therapy, or a combinationthereof. In some embodiments, the second cancer treatment regimencomprises a B cell receptor pathway inhibitor. In some embodiments, theB cell receptor pathway inhibitor is a CD79A inhibitor, a CD79Binhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3Kinhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or acombination thereof. In some embodiments, the second cancer treatmentregimen comprises an antibody, B cell receptor signaling inhibitor, aPI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, aradioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, ahistone deacetylase inhibitor, a protein kinase inhibitor, a hedgehoginhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1/2inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or acombination thereof. In some embodiments, the second cancer treatmentregimen comprises chlorambucil, ifosphamide, doxorubicin, mesalazine,thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine,fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab,dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab,bortezomib, pentostatin, endostatin, or a combination thereof. In someembodiments, the second cancer treatment regimen comprisescyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, andoptionally, rituximab. In some embodiments, the second cancer treatmentregimen comprises bendamustine, and rituximab. In some embodiments, thesecond cancer treatment regimen comprises fludarabine, cyclophosphamide,and rituximab. In some embodiments, the second cancer treatment regimencomprises cyclophosphamide, vincristine, and prednisone, and optionally,rituximab. In some embodiments, the second cancer treatment regimencomprises etoposide, doxorubicin, vinristine, cyclophosphamide,prednisolone, and optionally, rituximab. In some embodiments, the secondcancer treatment regimen comprises dexamethasone and lenalidomide.

Disclosed herein, in certain embodiments, is a method for treatingdiffuse large B-cell lymphoma, activated B cell-like subtype(ABC-DLBCL), in an individual in need thereof, comprising: administeringto the individual an irreversible Btk inhibitor in an amount from 300mg/day up to, and including, 1000 mg/day. In some embodiments, themethod further comprises diagnosing the individual with diffuse largeB-cell lymphoma, activated B cell-like subtype (ABC-DLBCL), bydetermining the gene sequence of one or more biomarkers in a pluralityof lymphoid cells isolated from the diffuse large B-cell lymphoma. Insome embodiments, the irreversible Btk inhibitor is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.In some embodiments, the ABC-DLBCL is characterized by a CD79B mutation.In some embodiments, the CD79B mutation is a mutation of theimmunoreceptor tyrosine-based activation motif (ITAM) signaling module.In some embodiments, the CD79B mutation is a missense mutation of thefirst immunoreceptor tyrosine-based activation motif (ITAM) tyrosine. Insome embodiments, the CD79B mutation increases surface BCR expressionand attenuates Lyn kinase activity. In some embodiments, the ABC-DLBCLis characterized by a CD79A mutation. In some embodiments, the CD79Amutation is in the immunoreceptor tyrosine-based activation motif (ITAM)signaling module. In some embodiments, the CD79A mutation is asplice-donor-site mutation of the immunoreceptor tyrosine-basedactivation motif (ITAM) signaling module. In some embodiments, the CD79Amutation deletes the immunoreceptor tyrosine-based activation motif(ITAM) signaling module. In some embodiments, the ABC-DLBCL ischaracterized by a mutation in MyD88, A20, or a combination thereof. Insome embodiments, the MyD88 mutation is the amino acid substitutionL265P in the MYD88 Toll/IL-1 receptor (TIR) domain. In some embodiments,the amount of the irreversible Btk inhibitor is from 420 mg/day up to,and including, 840 mg/day. In some embodiments, the amount of theirreversible Btk inhibitor is about 420 mg/day, about 560 mg/day, orabout 840 mg/day. In some embodiments, the amount of the irreversibleBtk inhibitor is about 420 mg/day. In some embodiments, the AUC₀₋₂₄ ofthe Btk inhibitor is between about 150 and about 3500 ng*h/mL. In someembodiments, the AUC₀₋₂₄ of the Btk inhibitor is between about 500 andabout 1100 ng*h/mL. In some embodiments, the irreversible Btk inhibitoris administered orally. In some embodiments, the irreversible Btkinhibitor is administered daily until disease progression, unacceptabletoxicity, or individual choice. In some embodiments, the irreversibleBtk inhibitor is administered every other day until disease progression,unacceptable toxicity, or individual choice. In some embodiments, theirreversible Btk inhibitor is a front line therapy, second line therapy,third line therapy, fourth line therapy, fifth line therapy, or sixthline therapy. In some embodiments, the irreversible Btk inhibitor treatsa refractory hematological malignancy. In some embodiments, theirreversible Btk inhibitor is a maintenance therapy. In someembodiments, the method further comprises administering at least oneadditional cancer treatment regimen. In some embodiments, the additionalcancer treatment regimen comprises a chemotherapeutic agent, animmunotherapeutic agent, a steroid, radiation therapy, a targetedtherapy, or a combination thereof. In some embodiments, the secondcancer treatment regimen comprises an antibody, B cell receptorsignaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTORinhibitor, a radioimmunotherapeutic, in certain embodiments is adamaging agent, a proteosome inhibitor, a histone deacetylase inhibitor,a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, atelomerase inhibitor, a Jak1/2 inhibitor, a protease inhibitor, a PKCinhibitor, a PARP inhibitor, or a combination thereof.

Disclosed herein, in certain embodiments, is a method of determining acancer treatment regimen for an individual with a hematologicalmalignancy, comprising: (a) administering to the individual an amount ofan irreversible Btk inhibitor sufficient to mobilize a plurality ofcells from the malignancy; (b) analyzing the mobilized plurality ofcells; and (c) selecting a cancer treatment regimen. In someembodiments, the cancer treatment regimen comprises a chemotherapeuticagent, a steroid, an immunotherapeutic agent, a targeted therapy, or acombination thereof. In some embodiments, the second cancer treatmentregimen comprises a B cell receptor pathway inhibitor. In someembodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor,a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, aPI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, ora combination thereof. In some embodiments, the cancer treatment regimencomprises a B cell receptor pathway inhibitor. In some embodiments, thecancer treatment regimen comprises a CD79A inhibitor, a CD79B inhibitor,a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, aBlnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combinationthereof. In some embodiments, the cancer treatment regimen comprises anantibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAPinhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damagingagent, a proteosome inhibitor, a histone deacetylase inhibitor, aprotein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, atelomerase inhibitor, a Jak1/2 inhibitor, a protease inhibitor, a PKCinhibitor, a PARP inhibitor, or a combination thereof. In someembodiments, the cancer treatment regimen comprises chlorambucil,ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide,temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel,docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101,ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or acombination thereof. In some embodiments, the cancer treatment regimencomprises cyclophosphamide, hydroxydaunorubicin, vincristine, andprednisone, and optionally, rituximab. In some embodiments, the cancertreatment regimen comprises bendamustine, and rituximab. In someembodiments, the cancer treatment regimen comprises fludarabine,cyclophosphamide, and rituximab. In some embodiments, the cancertreatment regimen comprises cyclophosphamide, vincristine, andprednisone, and optionally, rituximab. In some embodiments, the cancertreatment regimen comprises etoposide, doxorubicin, vinristine,cyclophosphamide, prednisolone, and optionally, rituximab. In someembodiments, the cancer treatment regimen comprises dexamethasone andlenalidomide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the role of Btk activity in a number of processes in aCLL cell that contribute to the pathogenesis of the disease

FIG. 2 presents the absolute lyphocyte count during the course oftreatment with an irreversible Btk inhibitor for an individual with CLL.

FIG. 3 presents change in the sum of the product of the diameters oflymph node (LN) in patients with CLL and SLL who are treated with anirreversible Btk inhibitor.

FIG. 4 depicts LN response in patient suffering from CLL. Left paneldepicts LN prior to treatment with an irreversible Btk inhibitor andRight panel depicts LN post-treatment with an irreversible Btkinhibitor.

FIG. 5 depicts the effect of an irreversible Btk inhibitor on LN diseaseburden and lymphocytosis over time in the patients suffering with CLLand/or SLL.

FIG. 6 depicts adverse effects in patients treated with an irreversibleBtk inhibitor. Grades 1-4 represent severity of effects with 1representing very mild to 4 representing extreme discomfort.

FIG. 7 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to individuals with follicularlymphoma who achieved complete or partial response (CR/PR). The Y Axisshows the Absolute Lymphocyte Counts (ALC) at each time point by cyclenumber and day in the X axis. All Patients (except Pt 32009) weretreated on schedule of 4 weeks on treatment followed by one week off.Thus, day 1 of each cycle follows one week off drug for these patients.Note the increases of ALC during most cycles of most patients, and thefall of ALC at the beginning of subsequent cycles. This pattern is oftenblunted in later cycles as patients responded to treatment. Patient32009 received treatment without interruption and did not show thiscyclic pattern, but did show an increase at Cycle 1, day 15, and gradualincreases during Cycles 2 to 5.

FIG. 8 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to individuals with follicularlymphoma who had Stable Disease (SD) during treatment. The Y Axis showsthe Absolute Lymphocyte Counts (ALC) at each time point by cycle numberand day in the X axis. All Patients were treated on schedule of 4 weekson treatment followed by one week off. Thus, day 1 of each cycle followsone week off drug for these patients. Note the gradual increase of bloodALC mobilization of Patient 32004, who initially was stable but laterhad Progressive Disease (PD).

FIG. 9 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to PD individuals with follicularlymphoma. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at eachtime point by cycle number and day in the X axis. All Patients except38010 were treated on schedule of 4 weeks on treatment followed by oneweek off. Thus, day 1 of each cycle follows one week off drug for thesepatients. Note lack of mobilization, especially patients 38010 and32001. Patient 323001 had limited treatment before being taken offstudy. The lymphocyte response suggests that this patient might hadresponded if it had been possible to stay on treatment longer.

FIG. 10 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to PR and SD individuals with DLBCL.The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time pointby cycle number and day in the X axis. Patient 38011 was treated onschedule of 4 weeks on treatment followed by one week off. Thus, day 1of each cycle follows one week off drug for this patient. Patients 38008and 324001 were treated with continuous daily doses.

FIG. 11 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to PD individuals with DLBCL. The YAxis shows the Absolute Lymphocyte Counts (ALC) at each time point bycycle number and day in the X axis. All Patients were treated onschedule of 4 weeks on treatment followed by one week off. Thus, day 1of each cycle follows one week off drug for these patients. Note lack ofmobilization for 3 of the 4 patients. Patient 32002 received only onecycle of treatment.

FIG. 12 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayafter administering a Btk inhibitor to individuals with mantle celllymphoma. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at eachtime point by cycle number and day in the X axis. Patients 32006, 38003,and 38004 were treated on schedule of 4 weeks on treatment followed byone week off. Thus, day 1 of each cycle follows one week off drug forthese patients. The other patients were treated with continuous dailydosing. Note that the patient with initial PD (32014) failed to showmobilization.

FIG. 13 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Dayfor after administering a Btk inhibitor to the individuals with mantlecell lymphoma shown in FIG. 12. The axis has been changed, as comparedto FIG. 12, to demonstrate low amplitude fluctuations. Note that allresponding patients showed some degree of mobilization.

FIG. 14 demonstrates that lymphocyte mobilization, specifically B Celltype, consistent with lymphoma cells, decreases as disease responds.Patient 32007, Cohort 4, had follicular lymphoma, grade 3, whichgradually regressed from SD to CR. Although the changes of ALC in thiscase are not dramatic, the B cell fraction is undergoing characteristiccyclic increases in response to treatment with a Btk inhibitor. Alsonote the decreasing cycle by cycle magnitude of shifts consistent withcumulative disease control.

FIG. 15 demonstrates that there is increased B Cell mobilization withdisease progression. Patient 32004, Cohort 2, had follicular lymphoma,grade 1, which progressed from SD initially to PD following Cycle 6.

FIG. 16 depicts early mobilization and eventual decrease of a CD45^(DIM)B cell subpopulation in responding mantle cell lymphoma patient 200-005.This subpopulation has a typical MCL immunophenotype (CD45^(DIM)) and isdifferent than that of normal lymphocytes.

FIG. 17 depicts abnormal high light scatter CD19⁺ cells mobilizing andthen regressing in CR DLBCL Pt 324001. These CD45⁺ cells with lightscatter (SSC-H) in the upper panels were gated upon and their CD3 vsCD19 staining displayed in the lower panels. Here the putative malignantcells were “hidden” in the large MNC window normally defining monocytes.The sequence of mobilization followed by response is similar to otherexamples.

FIG. 18 presents the responses for a clinical trial involvingadministering a Btk inhibitor to elderly patients with CLL or SLL, whoare naïve for drug intervention. Individuals were administered 420mg/day of a Btk inhibitor.

FIG. 19 presents the responses for a clinical trial involvingadministering a Btk inhibitor to R/R patients with CLL or SLL.Individuals were administered 420 mg/day of a Btk inhibitor.

FIG. 20 presents the responses for a clinical trial involvingadministering a Btk inhibitor to individuals with high risk CLL.

FIG. 21 presents the response over time for a clinical trial involvingadministering a Btk inhibitor to individuals with CLL or SLL.

FIG. 22 presents the best responses for all patients in a clinical trialinvolving administering a Btk inhibitor to individuals with CLL or SLL.

FIG. 23 presents the best responses for abstract patients in a clinicaltrial involving administering a Btk inhibitor to individuals with CLL orSLL.

FIG. 24 presents the best response by prognostic factor in CLL or SLLpatients involved in a clinical trial involving administering a Btkinhibitor.

FIG. 25 presents initial (Cycle 2) response assessment and best response(420 mg Cohorts) in CLL or SLL patients involved in a clinical trialinvolving administering a Btk inhibitor.

FIG. 26 presents initial (Cycle 2) response assessment by dose inrelapsed/refractory CLL or SLL patients involved in a clinical trialinvolving administering a Btk inhibitor.

FIG. 27 presents improvements in hematological parameters in CLL or SLLpatients involved in a clinical trial involving administering a Btkinhibitor.

FIG. 28 present data showing the results of a combination of a Btkinhibitor and Carboplatin or Velcade in DoHH2 cells.

FIG. 29 present data showing the results of a combination of a Btkinhibitor and Dexamethasone or Lenalidomide in DoHH2 cells.

FIG. 30 present data showing the results of a combination of a Btkinhibitor and Temsirolimus or R406 in DoHH2 cells.

FIG. 31 present data showing the results of a combination of a Btkinhibitor and Gemcitabine or Doxorubicin in DoHH2 cells.

FIG. 32 present data showing the results of a combination of a Btkinhibitor and Cal-101 in TMD8 cells.

FIG. 33 present data showing the results of a combination of a Btkinhibitor and R406 in TMD8 cells.

FIG. 34 present data showing the results of a combination of a Btkinhibitor and vincristine in TMD8 cells.

FIG. 35 present data showing the results of a combination of a Btkinhibitor and doxorubicin in TMD8 cells.

FIG. 36 present data showing the results of a combination of a Btkinhibitor and lenolidomide in TMD8 cells.

FIG. 37 present data showing the results of a combination of a Btkinhibitor and velcade in TMD8 cells.

FIG. 38 present data showing the results of a combination of a Btkinhibitor and Fludarabine in TMD8 cells.

FIG. 39 present data showing the results of a combination of a Btkinhibitor and taxol in TMD8 cells.

DETAILED DESCRIPTION OF THE INVENTION

There is currently a need for methods of treating (including,diagnosing) hematological malignancies, including relapsed andrefractory B cell malignancies, and ABC-DLBCL. The present applicationis based, in part, on the unexpected discovery that Btk inhibitorsinduce mobilization (or, in some cases, lymphocytosis) of lymphoid cellsin solid hematological malignancies. Mobilization of the lymphoid cellsincreases their exposure to additional cancer treatment regimens andtheir availability for biomarker screening. The inventors have alsofound that Btk inhibitors are useful for treating relapsed andrefractory malignancies and ABC-DLBCL.

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells.Disclosed herein, in certain embodiments, is a method for treatingdiffuse large B-cell lymphoma, activated B cell-like subtype(ABC-DLBCL), in an individual in need thereof, comprising: administeringto the individual an irreversible Btk inhibitor in an amount from 300mg/day up to, and including, 1000 mg/day. Further disclosed herein, incertain embodiments, is a method for treating relapsed or refractorynon-Hodgkin's lymphoma in an individual in need thereof, comprising:administering to the individual a therapeutically-effective amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.

CERTAIN TERMINOLOGY

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. In the event that thereare a plurality of definitions for terms herein, those in this sectionprevail. Where reference is made to a URL or other such identifier oraddress, it is understood that such identifiers can change andparticular information on the internet can come and go, but equivalentinformation can be found by searching the internet. Reference theretoevidences the availability and public dissemination of such information.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, but not limited to, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Definition of standard chemistry terms may be found in reference works,including Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4^(TH) ED.”Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwiseindicated, conventional methods of mass spectroscopy, NMR, HPLC, proteinchemistry, biochemistry, recombinant DNA techniques and pharmacology,within the skill of the art are employed. Unless specific definitionsare provided, the nomenclature employed in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those known in the art. Standard techniques can be used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients. Standardtechniques can be used for recombinant DNA, oligonucleotide synthesis,and tissue culture and transformation (e.g., electroporation,lipofection). Reactions and purification techniques can be performede.g., using kits of manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures can be generally performed of conventional methods wellknown in the art and as described in various general and more specificreferences that are cited and discussed throughout the presentspecification.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods and compositions described herein,which will be limited only by the appended claims.

All publications and patents mentioned herein are incorporated herein byreference in their entirety for the purpose of describing anddisclosing, for example, the constructs and methodologies that aredescribed in the publications, which might be used in connection withthe methods, compositions and compounds described herein. Thepublications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors described herein arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety may be a “saturated alkyl” group, which means that it does notcontain any alkene or alkyne moieties. The alkyl moiety may also be an“unsaturated alkyl” moiety, which means that it contains at least onealkene or alkyne moiety. An “alkene” moiety refers to a group that hasat least one carbon-carbon double bond, and an “alkyne” moiety refers toa group that has at least one carbon-carbon triple bond. The alkylmoiety, whether saturated or unsaturated, may be branched, straightchain, or cyclic. Depending on the structure, an alkyl group can be amonoradical or a diradical (i.e., an alkylene group). The alkyl groupcould also be a “lower alkyl” having 1 to 6 carbon atoms.

As used herein, C₁-C_(x) includes C₁-C₂, C₁-C₃ . . . C₁-C_(x).

The “alkyl” moiety may have 1 to 10 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 10” refers to each integer inthe given range; e.g., “1 to 10 carbon atoms” means that the alkyl groupmay have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to andincluding 10 carbon atoms, although the present definition also coversthe occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group of the compounds described herein may bedesignated as “C₁-C₄ alkyl” or similar designations. By way of exampleonly, “C₁-C₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from among methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Thus C₁-C₄ alkyl includes C₁-C₂ alkyl and C₁-C₃ alkyl. Alkyl groups canbe substituted or unsubstituted. Typical alkyl groups include, but arein no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like.

As used herein, the term “non-cyclic alkyl” refers to an alkyl that isnot cyclic (i.e., a straight or branched chain containing at least onecarbon atom). Non-cyclic alkyls can be fully saturated or can containnon-cyclic alkenes and/or alkynes. Non-cyclic alkyls can be optionallysubstituted.

The term “alkenyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a double bond that is not part of anaromatic group. That is, an alkenyl group begins with the atoms—C(R)═C(R)—R, wherein R refers to the remaining portions of the alkenylgroup, which may be the same or different. The alkenyl moiety may bebranched, straight chain, or cyclic (in which case, it would also beknown as a “cycloalkenyl” group). Depending on the structure, an alkenylgroup can be a monoradical or a diradical (i.e., an alkenylene group).Alkenyl groups can be optionally substituted. Non-limiting examples ofan alkenyl group include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃, —C(CH₃)═CHCH₃.Alkenylene groups include, but are not limited to, —CH═CH—, —C(CH₃)═CH—,—CH═CHCH₂—, —CH═CHCH₂CH₂— and —C(CH₃)═CHCH₂—. Alkenyl groups could have2 to 10 carbons. The alkenyl group could also be a “lower alkenyl”having 2 to 6 carbon atoms.

The term “alkynyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a triple bond. That is, an alkynylgroup begins with the atoms —C≡C—R, wherein R refers to the remainingportions of the alkynyl group, which may be the same or different. The“R” portion of the alkynyl moiety may be branched, straight chain, orcyclic. Depending on the structure, an alkynyl group can be amonoradical or a diradical (i.e., an alkynylene group). Alkynyl groupscan be optionally substituted. Non-limiting examples of an alkynyl groupinclude, but are not limited to, —C≡CH, —C≡CCH₃, —C≡CCH₂CH₃, —C≡C—, and—C≡CCH₂—. Alkynyl groups can have 2 to 10 carbons. The alkynyl groupcould also be a “lower alkynyl” having 2 to 6 carbon atoms.

An “alkoxy” group refers to a (alkyl)O— group, where alkyl is as definedherein.

“Hydroxyalkyl” refers to an alkyl radical, as defined herein,substituted with at least one hydroxy group. Non-limiting examples of ahydroxyalkyl include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl.

“Alkoxyalkyl” refers to an alkyl radical, as defined herein, substitutedwith an alkoxy group, as defined herein.

An “alkenyloxy” group refers to a (alkenyl)O— group, where alkenyl is asdefined herein.

The term “alkylamine” refers to the —N(alkyl)_(x)H_(y) group, where xand y are selected from among x=1, y=1 and x=2, y=0. When x=2, the alkylgroups, taken together with the N atom to which they are attached, canoptionally form a cyclic ring system.

“Alkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine, as defined herein.

An “amide” is a chemical moiety with the formula —C(O)NHR or —NHC(O)R,where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) and heteroalicyclic (bonded through aring carbon). An amide moiety may form a linkage between an amino acidor a peptide molecule and a compound described herein, thereby forming aprodrug. Any amine, or carboxyl side chain on the compounds describedherein can be amidified. The procedures and specific groups to make suchamides are known to those of skill in the art and can readily be foundin reference sources such as Greene and Wuts, Protective Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York, N.Y., 1999,which is incorporated herein by reference in its entirety.

The term “ester” refers to a chemical moiety with formula —COOR, where Ris selected from among alkyl, cycloalkyl, aryl, heteroaryl (bondedthrough a ring carbon) and heteroalicyclic (bonded through a ringcarbon). Any hydroxy, or carboxyl side chain on the compounds describedherein can be esterified. The procedures and specific groups to makesuch esters are known to those of skill in the art and can readily befound in reference sources such as Greene and Wuts, Protective Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York, N.Y., 1999,which is incorporated herein by reference in its entirety.

As used herein, the term “ring” refers to any covalently closedstructure. Rings include, for example, carbocycles (e.g., aryls andcycloalkyls), heterocycles (e.g., heteroaryls and non-aromaticheterocycles), aromatics (e.g. aryls and heteroaryls), and non-aromatics(e.g., cycloalkyls and non-aromatic heterocycles). Rings can beoptionally substituted. Rings can be monocyclic or polycyclic.

As used herein, the term “ring system” refers to one, or more than onering.

The term “membered ring” can embrace any cyclic structure. The term“membered” is meant to denote the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

The term “fused” refers to structures in which two or more rings shareone or more bonds.

The term “carbocyclic” or “carbocycle” refers to a ring wherein each ofthe atoms forming the ring is a carbon atom. Carbocycle includes aryland cycloalkyl. The term thus distinguishes carbocycle from heterocycle(“heterocyclic”) in which the ring backbone contains at least one atomwhich is different from carbon (i.e a heteroatom). Heterocycle includesheteroaryl and heterocycloalkyl. Carbocycles and heterocycles can beoptionally substituted.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2 π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, or morethan nine atoms. Aromatics can be optionally substituted. The term“aromatic” includes both carbocyclic aryl (e.g., phenyl) andheterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings can be formedby five, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups can be optionally substituted. Examples of aryl groups include,but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl,fluorenyl, and indenyl. Depending on the structure, an aryl group can bea monoradical or a diradical (i.e., an arylene group).

An “aryloxy” group refers to an (aryl)O— group, where aryl is as definedherein.

“Aralkyl” means an alkyl radical, as defined herein, substituted with anaryl group. Non-limiting aralkyl groups include, benzyl, phenethyl, andthe like.

“Aralkenyl” means an alkenyl radical, as defined herein, substitutedwith an aryl group, as defined herein.

The term “cycloalkyl” refers to a monocyclic or polycyclic radical thatcontains only carbon and hydrogen, and may be saturated, partiallyunsaturated, or fully unsaturated. Cycloalkyl groups include groupshaving from 3 to 10 ring atoms. Illustrative examples of cycloalkylgroups include the following moieties:

and the like. Depending on the structure, a cycloalkyl group can be amonoradical or a diradical (e.g., an cycloalkylene group). Thecycloalkyl group could also be a “lower cycloalkyl” having 3 to 8 carbonatoms.

“Cycloalkylalkyl” means an alkyl radical, as defined herein, substitutedwith a cycloalkyl group. Non-limiting cycloalkylalkyl groups includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, and the like.

The term “heterocycle” refers to heteroaromatic and heteroalicyclicgroups containing one to four heteroatoms each selected from O, S and N,wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the ring of said group does notcontain two adjacent O or S atoms. Herein, whenever the number of carbonatoms in a heterocycle is indicated (e.g., C₁-C₆ heterocycle), at leastone other atom (the heteroatom) must be present in the ring.Designations such as “C₁-C₆ heterocycle” refer only to the number ofcarbon atoms in the ring and do not refer to the total number of atomsin the ring. It is understood that the heterocylic ring can haveadditional heteroatoms in the ring. Designations such as “4-6 memberedheterocycle” refer to the total number of atoms that are contained inthe ring (i.e., a four, five, or six membered ring, in which at leastone atom is a carbon atom, at least one atom is a heteroatom and theremaining two to four atoms are either carbon atoms or heteroatoms). Inheterocycles that have two or more heteroatoms, those two or moreheteroatoms can be the same or different from one another. Heterocyclescan be optionally substituted. Binding to a heterocycle can be at aheteroatom or via a carbon atom. Non-aromatic heterocyclic groupsinclude groups having only 4 atoms in their ring system, but aromaticheterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a4-membered heterocyclic group is azetidinyl (derived from azetidine). Anexample of a 5-membered heterocyclic group is thiazolyl. An example of a6-membered heterocyclic group is pyridyl, and an example of a10-membered heterocyclic group is quinolinyl. Examples of non-aromaticheterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups, as derived from the groups listedabove, may be C-attached or N-attached where such is possible. Forinstance, a group derived from pyrrole may be pyrrol-1-yl (N-attached)or pyrrol-3-yl (C-attached). Further, a group derived from imidazole maybe imidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl,imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groupsinclude benzo-fused ring systems and ring systems substituted with oneor two oxo (═O) moieties such as pyrrolidin-2-one. Depending on thestructure, a heterocycle group can be a monoradical or a diradical(i.e., a heterocyclene group).

The terms “heteroaryl” or, alternatively, “heteroaromatic” refers to anaryl group that includes one or more ring heteroatoms selected fromnitrogen, oxygen and sulfur. An N-containing “heteroaromatic” or“heteroaryl” moiety refers to an aromatic group in which at least one ofthe skeletal atoms of the ring is a nitrogen atom. Illustrative examplesof heteroaryl groups include the following moieties:

and the like. Depending on the structure, a heteroaryl group can be amonoradical or a diradical (i.e., a heteroarylene group).

As used herein, the term “non-aromatic heterocycle”, “heterocycloalkyl”or “heteroalicyclic” refers to a non-aromatic ring wherein one or moreatoms forming the ring is a heteroatom. A “non-aromatic heterocycle” or“heterocycloalkyl” group refers to a cycloalkyl group that includes atleast one heteroatom selected from nitrogen, oxygen and sulfur. Theradicals may be fused with an aryl or heteroaryl. Heterocycloalkyl ringscan be formed by three, four, five, six, seven, eight, nine, or morethan nine atoms. Heterocycloalkyl rings can be optionally substituted.In certain embodiments, non-aromatic heterocycles contain one or morecarbonyl or thiocarbonyl groups such as, for example, oxo- andthio-containing groups. Examples of heterocycloalkyls include, but arenot limited to, lactams, lactones, cyclic imides, cyclic thioimides,cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran,piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane,piperazine, 1,3-oxathiane, 1,4-oxathiin, 1,4-oxathiane,tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide,barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin,dihydrouracil, morpholine, trioxane, hexahydro-1,3,5-triazine,tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine,pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline,imidazolidine, 1,3-dioxole, 1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane,isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone,thiazoline, thiazolidine, and 1,3-oxathiolane. Illustrative examples ofheterocycloalkyl groups, also referred to as non-aromatic heterocycles,include:

and the like. The term heteroalicyclic also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Depending on the structure, aheterocycloalkyl group can be a monoradical or a diradical (i.e., aheterocycloalkylene group).

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo and iodo.

The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl” and “haloalkoxy”include alkyl, alkenyl, alkynyl and alkoxy structures in which at leastone hydrogen is replaced with a halogen atom. In certain embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are all the same as one another. In other embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are not all the same as one another.

The term “fluoroalkyl,” as used herein, refers to alkyl group in whichat least one hydrogen is replaced with a fluorine atom. Examples offluoroalkyl groups include, but are not limited to, —CF₃, —CH₂CF₃,—CF₂CF₃, —CH₂CH₂CF₃ and the like.

As used herein, the terms “heteroalkyl” “heteroalkenyl” and“heteroalkynyl” include optionally substituted alkyl, alkenyl andalkynyl radicals in which one or more skeletal chain atoms is aheteroatom, e.g., oxygen, nitrogen, sulfur, silicon, phosphorus orcombinations thereof. The heteroatom(s) may be placed at any interiorposition of the heteroalkyl group or at the position at which theheteroalkyl group is attached to the remainder of the molecule. Examplesinclude, but are not limited to, —CH₂—O—CH₃, —CH₂—CH₂—O—CH₃,—CH₂—NH—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—N(CH₃)—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. In addition, up to two heteroatoms may beconsecutive, such as, by way of example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

The term “heteroatom” refers to an atom other than carbon or hydrogen.Heteroatoms are typically independently selected from among oxygen,sulfur, nitrogen, silicon and phosphorus, but are not limited to theseatoms. In embodiments in which two or more heteroatoms are present, thetwo or more heteroatoms can all be the same as one another, or some orall of the two or more heteroatoms can each be different from theothers.

The term “bond” or “single bond” refers to a chemical bond between twoatoms, or two moieties when the atoms joined by the bond are consideredto be part of larger substructure.

An “isocyanato” group refers to a —NCO group.

An “isothiocyanato” group refers to a —NCS group.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

A “sulfinyl” group refers to a —S(═O)—R.

A “sulfonyl” group refers to a —S(═O)₂—R.

A “thioalkoxy” or “alkylthio” group refers to a —S-alkyl group.

A “alkylthioalkyl” group refers to an alkyl group substituted with a—S-alkyl group.

As used herein, the term “O-carboxy” or “acyloxy” refers to a group offormula RC(═O)O—.

“Carboxy” means a —C(O)OH radical.

As used herein, the term “acetyl” refers to a group of formula—C(═O)CH₃.

“Acyl” refers to the group —C(O)R.

As used herein, the term “trihalomethanesulfonyl” refers to a group offormula X₃CS(═O)₂— where X is a halogen.

As used herein, the term “cyano” refers to a group of formula —CN.

“Cyanoalkyl” means an alkyl radical, as defined herein, substituted withat least one cyano group.

As used herein, the term “N-sulfonamido” or “sulfonylamino” refers to agroup of formula RS(═O)₂NH—.

As used herein, the term “O-carbamyl” refers to a group of formula—OC(═O)NR₂.

As used herein, the term “N-carbamyl” refers to a group of formulaROC(═O)NH—.

As used herein, the term “0-thiocarbamyl” refers to a group of formula—OC(═S)NR2.

As used herein, the term “N-thiocarbamyl” refers to a group of formulaROC(═S)NH—.

As used herein, the term “C-amido” refers to a group of formula—C(═O)NR2.

“Aminocarbonyl” refers to a —CONH2 radical.

As used herein, the term “N-amido” refers to a group of formulaRC(═O)NH—.

As used herein, the substituent “R” appearing by itself and without anumber designation refers to a substituent selected from among fromalkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andnon-aromatic heterocycle (bonded through a ring carbon).

The term “optionally substituted” or “substituted” means that thereferenced group may be substituted with one or more additional group(s)individually and independently selected from alkyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio,arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone,cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, including mono-and di-substituted amino groups, and the protected derivatives thereof.By way of example an optional substituents may be L_(s)R_(s), whereineach L_(s) is independently selected from a bond, —O—, —C(═O)—, —S—,—S(═O)—, —S(═O)₂—, —NH—, —NHC(O)—, —C(O)NH—, S(═O)₂NH—, —NHS(═O)₂,—OC(O)NH—, —NHC(O)O—, -(substituted or unsubstituted C₁-C₆ alkyl), or-(substituted or unsubstituted C₂-C₆ alkenyl); and each R_(s) isindependently selected from H, (substituted or unsubstitutedC₁-C₄alkyl), (substituted or unsubstituted C₃-C₆cycloalkyl), heteroaryl,or heteroalkyl. The protecting groups that may form the protectivederivatives of the above substituents are known to those of skill in theart and may be found in references such as Greene and Wuts, above.

The term “Michael acceptor moiety” refers to a functional group that canparticipate in a Michael reaction, wherein a new covalent bond is formedbetween a portion of the Michael acceptor moiety and the donor moiety.The Michael acceptor moiety is an electrophile and the “donor moiety” isa nucleophile.

The term “nucleophile” or “nucleophilic” refers to an electron richcompound, or moiety thereof. An example of a nucleophile includes, butin no way is limited to, a cysteine residue of a molecule, such as, forexample Cys 481 of Btk.

The term “electrophile”, or “electrophilic” refers to an electron pooror electron deficient molecule, or moiety thereof. Examples ofelectrophiles include, but in no way are limited to, Micheal acceptormoieties.

The term “acceptable” or “pharmaceutically acceptable”, with respect toa formulation, composition or ingredient, as used herein, means havingno persistent detrimental effect on the general health of the subjectbeing treated or does not abrogate the biological activity or propertiesof the compound, and is relatively nontoxic.

“B-cell lymphoproliferative disorders (BCLD) biomarkers”, as usedherein, refer to any biological molecule (found either in blood, otherbody fluids, or tissues) or any chromosomal abnormality that is a signof a BCLD-related condition or disease.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic,” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, B-cell lymphoproliferative disorders (BCLDs), such as lymphoma andleukemia, and solid tumors. By “B cell-related cancer” or “cancer ofB-cell lineage” is intended any type of cancer in which the dysregulatedor unregulated cell growth is associated with B cells.

By “refractory” in the context of a cancer is intended the particularcancer is resistant to, or non-responsive to, therapy with a particulartherapeutic agent. A cancer can be refractory to therapy with aparticular therapeutic agent either from the onset of treatment with theparticular therapeutic agent (i.e., non-responsive to initial exposureto the therapeutic agent), or as a result of developing resistance tothe therapeutic agent, either over the course of a first treatmentperiod with the therapeutic agent or during a subsequent treatmentperiod with the therapeutic agent.

By “agonist activity” is intended that a substance functions as anagonist. An agonist combines with a receptor on a cell and initiates areaction or activity that is similar to or the same as that initiated bythe receptor's natural ligand.

By “antagonist activity” is intended that the substance functions as anantagonist. An antagonist of Btk prevents or reduces induction of any ofthe responses mediated by Btk.

By “significant” agonist activity is intended an agonist activity of atleast 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or100% greater than the agonist activity induced by a neutral substance ornegative control as measured in an assay of a B cell response.Preferably, “significant” agonist activity is an agonist activity thatis at least 2-fold greater or at least 3-fold greater than the agonistactivity induced by a neutral substance or negative control as measuredin an assay of a B cell response. Thus, for example, where the B cellresponse of interest is B cell proliferation, “significant” agonistactivity would be induction of a level of B cell proliferation that isat least 2-fold greater or at least 3-fold greater than the level of Bcell proliferation induced by a neutral substance or negative control.

A substance “free of significant agonist activity” would exhibit anagonist activity of not more than about 25% greater than the agonistactivity induced by a neutral substance or negative control, preferablynot more than about 20% greater, 15% greater, 10% greater, 5% greater,1% greater, 0.5% greater, or even not more than about 0.1% greater thanthe agonist activity induced by a neutral substance or negative controlas measured in an assay of a B cell response.

In some embodiments, the Btk inhibitor therapeutic agent is anantagonist anti-Btk antibody. Such antibodies are free of significantagonist activity as noted above when bound to a Btk antigen in a humancell. In one embodiment of the invention, the antagonist anti-Btkantibody is free of significant agonist activity in one cellularresponse. In another embodiment of the invention, the antagonistanti-Btk antibody is free of significant agonist activity in assays ofmore than one cellular response (e.g., proliferation anddifferentiation, or proliferation, differentiation, and, for B cells,antibody production).

By “Btk-mediated signaling” it is intended any of the biologicalactivities that are dependent on, either directly or indirection, theactivity of Btk. Examples of Btk-mediated signaling are signals thatlead to proliferation and survival of Btk-expressing cells, andstimulation of one or more Btk-signaling pathways within Btk-expressingcells.

A Btk “signaling pathway” or “signal transduction pathway” is intendedto mean at least one biochemical reaction, or a group of biochemicalreactions, that results from the activity of Btk, and which generates asignal that, when transmitted through the signal pathway, leads toactivation of one or more downstream molecules in the signaling cascade.Signal transduction pathways involve a number of signal transductionmolecules that lead to transmission of a signal from the cell-surfaceacross the plasma membrane of a cell, and through one or more in aseries of signal transduction molecules, through the cytoplasm of thecell, and in some instances, into the cell's nucleus. Of particularinterest to the present invention are Btk signal transduction pathwayswhich ultimately regulate (either enhance or inhibit) the activation ofNF-κB via the NF-κB signaling pathway.

The methods of the present invention are directed to methods fortreating cancer that, in certain embodiments, utilize antibodies fordetermining the expression or presence of certain BCLD biomarkers inthese methods. The following terms and definitions apply to suchantibodies.

Antibodies” and “immunoglobulins” (Igs) are glycoproteins having thesame structural characteristics. The terms are used synonymously. Insome instances the antigen specificity of the immunoglobulin may beknown.

The term “antibody” is used in the broadest sense and covers fullyassembled antibodies, antibody fragments that can bind antigen (e.g.,Fab, F(ab′)₂, Fv, single chain antibodies, diabodies, antibody chimeras,hybrid antibodies, bispecific antibodies, humanized antibodies, and thelike), and recombinant peptides comprising the forgoing.

The terms “monoclonal antibody” and “mAb” as used herein refer to anantibody obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies.Variable regions confer antigen-binding specificity. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called complementaritydetermining regions (CDRs) or hypervariable regions, both in the lightchain and the heavy-chain variable domains. The more highly conservedportions of variable domains are celled in the framework (FR) regions.The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-pleated-sheet configuration, connectedby three CDRs, which form loops connecting, and in some cases formingpart of, the β-pleated-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen-binding siteof antibodies (see, Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I,pages 647-669). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as Fc receptor (FcR) binding, participation of theantibody in antibody-dependent cellular toxicity, initiation ofcomplement dependent cytotoxicity, and mast cell degranulation.

The term “hypervariable region,” when used herein, refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarily determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy-chain variabledomain; Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institute of Health,Bethesda, Md.) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chainvariable domain and (H1), 53-55 (H2), and 96-101 (13) in the heavy chainvariable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901-917).“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues, as herein deemed.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab, F(ab′)2, andFv fragments; diabodies; linear antibodies (Zapata et al. (1995) ProteinEng. 10:1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H1)) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain C_(H1) domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. Fab′ fragments are produced by reducing theF(ab′)2 fragment's heavy chain disulfide bridge. Other chemicalcouplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions. For example, humanIgG1 and IgG3 isotypes have ADCC (antibody dependent cell-mediatedcytotoxicity) activity.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody soas to generate a “labeled” antibody. The label may be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

The term “acceptable” or “pharmaceutically acceptable”, with respect toa formulation, composition or ingredient, as used herein, means havingno persistent detrimental effect on the general health of the subjectbeing treated or does not abrogate the biological activity or propertiesof the compound, and is relatively nontoxic.

As used herein, the term “agonist” refers to a compound, the presence ofwhich results in a biological activity of a protein that is the same asthe biological activity resulting from the presence of a naturallyoccurring ligand for the protein, such as, for example, Btk.

As used herein, the term “partial agonist” refers to a compound thepresence of which results in a biological activity of a protein that isof the same type as that resulting from the presence of a naturallyoccurring ligand for the protein, but of a lower magnitude.

As used herein, the term “antagonist” refers to a compound, the presenceof which results in a decrease in the magnitude of a biological activityof a protein. In certain embodiments, the presence of an antagonistresults in complete inhibition of a biological activity of a protein,such as, for example, Btk. In certain embodiments, an antagonist is aninhibitor.

The term “Bruton's tyrosine kinase (Btk),” as used herein, refers toBruton's tyrosine kinase from Homo sapiens, as disclosed in, e.g., U.S.Pat. No. 6,326,469 (GenBank Accession No. NP_000052).

The term “Bruton's tyrosine kinase homolog,” as used herein, refers toorthologs of Bruton's tyrosine kinase, e.g., the orthologs from mouse(GenBank Accession No. AAB47246), dog (GenBank Accession No.XP.sub.--549139), rat (GenBank Accession No. NP.sub.--001007799),chicken (GenBank Accession No. NP.sub.--989564), or zebra fish (GenBankAccession No. XP.sub.--698117), and fusion proteins of any of theforegoing that exhibit kinase activity towards one or more substrates ofBruton's tyrosine kinase (e.g. a peptide substrate having the amino acidsequence “AVLESEEELYSSARQ”) (SEQ ID NO: 1).

The terms “co-administration” or “combination therapy” and the like, asused herein, are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are administered by the same ordifferent route of administration or at the same or different time.

The term “effective amount,” as used herein, refers to a sufficientamount of a Btk inhibitory agent or a Btk inhibitor compound beingadministered which will result in an increase or appearance in the bloodof a subpopulation of lymphocytes (e.g., pharmaceutical debulking). Forexample, an “effective amount” for diagnostic and/or prognostic uses isthe amount of the composition including a compound as disclosed hereinrequired to provide a clinically significant decrease an increase orappearance in the blood of a subpopulation of lymphocytes without undueadverse side effects. An appropriate “effective amount” in anyindividual case may be determined using techniques, such as a doseescalation study.

The term “therapeutically effective amount,” as used herein, refers to asufficient amount of an agent or a compound being administered whichwill relieve to some extent one or more of the symptoms s B-celllymphoproliferative disorder (BCLD). The result can be reduction and/oralleviation of the signs, symptoms, or causes of BCLD, or any otherdesired alteration of a biological system. The term “therapeuticallyeffective amount” includes, for example, a prophylactically effectiveamount. An “effective amount” of a compound disclosed herein is anamount effective to achieve a desired pharmacologic effect ortherapeutic improvement without undue adverse side effects. It isunderstood that “an effect amount” or “a therapeutically effectiveamount” can vary from subject to subject, due to variation in metabolismof the compound of any of Formula (A), Formula (B), Formula (C), orFormula (D), age, weight, general condition of the subject, thecondition being treated, the severity of the condition being treated,and the judgment of the prescribing physician. By way of example only,therapeutically effective amounts may be determined by routineexperimentation, including but not limited to a dose escalation clinicaltrial.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. By way of example, “enhancing”the effect of therapeutic agents refers to the ability to increase orprolong, either in potency or duration, the effect of therapeutic agentson during treatment of a disease, disorder or condition. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of a therapeutic agent in the treatmentof a disease, disorder or condition. When used in a patient, amountseffective for this use will depend on the severity and course of thedisease, disorder or condition, previous therapy, the patient's healthstatus and response to the drugs, and the judgment of the treatingphysician.

The term “homologous cysteine,” as used herein refers to a cysteineresidue found with in a sequence position that is homologous to that ofcysteine 481 of Bruton's tyrosine kinase, as defined herein. Forexample, cysteine 482 is the homologous cysteine of the rat ortholog ofBruton's tyrosine kinase; cysteine 479 is the homologous cysteine of thechicken ortholog; and cysteine 481 is the homologous cysteine in thezebra fish ortholog. In another example, the homologous cysteine of TXK,a Tec kinase family member related to Bruton's tyrosine, is Cys 350. Seealso the sequence alignments of tyrosine kinases (TK) published on theworld wide web at kinase.com/human/kinome/phylogeny.html.

The term “identical,” as used herein, refers to two or more sequences orsubsequences which are the same. In addition, the term “substantiallyidentical,” as used herein, refers to two or more sequences which have apercentage of sequential units which are the same when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using comparison algorithms or by manualalignment and visual inspection. By way of example only, two or moresequences may be “substantially identical” if the sequential units areabout 60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. Suchpercentages to describe the “percent identity” of two or more sequences.The identity of a sequence can exist over a region that is at leastabout 75-100 sequential units in length, over a region that is about 50sequential units in length, or, where not specified, across the entiresequence. This definition also refers to the complement of a testsequence. By way of example only, two or more polypeptide sequences areidentical when the amino acid residues are the same, while two or morepolypeptide sequences are “substantially identical” if the amino acidresidues are about 60% identical, about 65% identical, about 70%identical, about 75% identical, about 80% identical, about 85%identical, about 90% identical, or about 95% identical over a specifiedregion. The identity can exist over a region that is at least about75-100 amino acids in length, over a region that is about 50 amino acidsin length, or, where not specified, across the entire sequence of apolypeptide sequence. In addition, by way of example only, two or morepolynucleotide sequences are identical when the nucleic acid residuesare the same, while two or more polynucleotide sequences are“substantially identical” if the nucleic acid residues are about 60%identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. The identitycan exist over a region that is at least about 75-100 nucleic acids inlength, over a region that is about 50 nucleic acids in length, or,where not specified, across the entire sequence of a polynucleotidesequence.

The terms “inhibits”, “inhibiting”, or “inhibitor” of a kinase, as usedherein, refer to inhibition of enzymatic phosphotransferase activity.

The term “irreversible inhibitor,” as used herein, refers to a compoundthat, upon contact with a target protein (e.g., a kinase) causes theformation of a new covalent bond with or within the protein, whereby oneor more of the target protein's biological activities (e.g.,phosphotransferase activity) is diminished or abolished notwithstandingthe subsequent presence or absence of the irreversible inhibitor.

The term “irreversible Btk inhibitor,” as used herein, refers to aninhibitor of Btk that can form a covalent bond with an amino acidresidue of Btk. In one embodiment, the irreversible inhibitor of Btk canform a covalent bond with a Cys residue of Btk; in particularembodiments, the irreversible inhibitor can form a covalent bond with aCys 481 residue (or a homolog thereof) of Btk or a cysteine residue inthe homologous corresponding position of another tyrosine kinase.

The term “isolated,” as used herein, refers to separating and removing acomponent of interest from components not of interest. Isolatedsubstances can be in either a dry or semi-dry state, or in solution,including but not limited to an aqueous solution. The isolated componentcan be in a homogeneous state or the isolated component can be a part ofa pharmaceutical composition that comprises additional pharmaceuticallyacceptable carriers and/or excipients. By way of example only, nucleicacids or proteins are “isolated” when such nucleic acids or proteins arefree of at least some of the cellular components with which it isassociated in the natural state, or that the nucleic acid or protein hasbeen concentrated to a level greater than the concentration of its invivo or in vitro production. Also, by way of example, a gene is isolatedwhen separated from open reading frames which flank the gene and encodea protein other than the gene of interest.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes, such as, oxidation reactions) by which aparticular substance is changed by an organism. Thus, enzymes mayproduce specific structural alterations to a compound. For example,cytochrome P450 catalyzes a variety of oxidative and reductive reactionswhile uridine diphosphate glucuronyl transferases catalyze the transferof an activated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulfhydryl groups. Furtherinformation on metabolism may be obtained from The Pharmacological Basisof Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of thecompounds disclosed herein can be identified either by administration ofcompounds to a host and analysis of tissue samples from the host, or byincubation of compounds with hepatic cells in vitro and analysis of theresulting compounds. Both methods are well known in the art. In someembodiments, metabolites of a compound are formed by oxidative processesand correspond to the corresponding hydroxy-containing compound. In someembodiments, a compound is metabolized to pharmacologically activemetabolites.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

As used herein, the term “modulator” refers to a compound that alters anactivity of a molecule. For example, a modulator can cause an increaseor decrease in the magnitude of a certain activity of a moleculecompared to the magnitude of the activity in the absence of themodulator. In certain embodiments, a modulator is an inhibitor, whichdecreases the magnitude of one or more activities of a molecule. Incertain embodiments, an inhibitor completely prevents one or moreactivities of a molecule. In certain embodiments, a modulator is anactivator, which increases the magnitude of at least one activity of amolecule. In certain embodiments the presence of a modulator results inan activity that does not occur in the absence of the modulator.

As used herein, the term “selective binding compound” refers to acompound that selectively binds to any portion of one or more targetproteins.

As used herein, the term “selectively binds” refers to the ability of aselective binding compound to bind to a target protein, such as, forexample, Btk, with greater affinity than it binds to a non-targetprotein. In certain embodiments, specific binding refers to binding to atarget with an affinity that is at least 10, 50, 100, 250, 500, 1000 ormore times greater than the affinity for a non-target.

As used herein, the term “selective modulator” refers to a compound thatselectively modulates a target activity relative to a non-targetactivity. In certain embodiments, specific modulator refers tomodulating a target activity at least 10, 50, 100, 250, 500, 1000 timesmore than a non-target activity.

The term “substantially purified,” as used herein, refers to a componentof interest that may be substantially or essentially free of othercomponents which normally accompany or interact with the component ofinterest prior to purification. By way of example only, a component ofinterest may be “substantially purified” when the preparation of thecomponent of interest contains less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating components. Thus, a“substantially purified” component of interest may have a purity levelof about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98%, about 99% or greater.

The term “subject” as used herein, refers to an animal which is theobject of treatment, observation or experiment. By way of example only,a subject may be, but is not limited to, a mammal including, but notlimited to, a human.

As used herein, the term “target activity” refers to a biologicalactivity capable of being modulated by a selective modulator. Certainexemplary target activities include, but are not limited to, bindingaffinity, signal transduction, enzymatic activity, tumor growth, effectson particular biomarkers related to B-cell lymphoproliferative disorderpathology.

As used herein, the term “target protein” refers to a molecule or aportion of a protein capable of being bound by a selective bindingcompound. In certain embodiments, a target protein is Btk.

The terms “treat,” “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating a disease or condition, or symptomsthereof; managing a disease or condition, or symptoms thereof;preventing additional symptoms; ameliorating or preventing theunderlying metabolic causes of symptoms; inhibiting the disease orcondition, e.g., arresting the development of the disease or condition;relieving the disease or condition; causing regression of the disease orcondition, relieving a condition caused by the disease or condition; orstopping the symptoms of the disease or condition. The terms “treat,”“treating” or “treatment”, include, but are not limited to, prophylacticand/or therapeutic treatments.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse, such as inhibition of Btk, in an assay that measures suchresponse.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

Hematological Malignancies

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, the hematological malignancy is CLL. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time. Insome embodiments, the hematological malignancy is a chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, or anon-CLL/SLL lymphoma. In some embodiments, the hematological malignancyis follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma,marginal zone lymphoma, Burkitt's lymphoma, non-Burkitt high grade Bcell lymphoma, or extranodal marginal zone B cell lymphoma. In someembodiments, the hematological malignancy is acute or chronicmyelogenous (or myeloid) leukemia, myelodysplastic syndrome, or acutelymphoblastic leukemia. In some embodiments, the hematologicalmalignancy is relapsed or refractory diffuse large B-cell lymphoma(DLBCL), relapsed or refractory mantle cell lymphoma, relapsed orrefractory follicular lymphoma, relapsed or refractory CLL; relapsed orrefractory SLL; relapsed or refractory multiple myeloma. In someembodiments, the hematological malignancy is a hematological malignancythat is classified as high-risk. In some embodiments, the hematologicalmalignancy is high risk CLL or high risk SLL.

B-cell lymphoproliferative disorders (BCLDs) are neoplasms of the bloodand encompass, inter alia, non-Hodgkin lymphoma, multiple myeloma, andleukemia. BCLDs can originate either in the lymphatic tissues (as in thecase of lymphoma) or in the bone marrow (as in the case of leukemia andmyeloma), and they all are involved with the uncontrolled growth oflymphocytes or white blood cells. There are many subtypes of BCLD, e.g.,chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Thedisease course and treatment of BCLD is dependent on the BCLD subtype;however, even within each subtype the clinical presentation, morphologicappearance, and response to therapy is heterogeneous.

Malignant lymphomas are neoplastic transformations of cells that residepredominantly within lymphoid tissues. Two groups of malignant lymphomasare Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL). Both types oflymphomas infiltrate reticuloendothelial tissues. However, they differin the neoplastic cell of origin, site of disease, presence of systemicsymptoms, and response to treatment (Freedman et al., “Non-Hodgkin'sLymphomas” Chapter 134, Cancer Medicine, (an approved publication of theAmerican Cancer Society, B.C. Decker Inc., Hamilton, Ontario, 2003).

Non-Hodgkin's Lymphomas

Disclosed herein, in certain embodiments, is a method for treating anon-Hodgkin's lymphoma in an individual in need thereof, comprising: (a)administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, the hematological malignancy is CLL. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

Further disclosed herein, in certain embodiments, is a method fortreating relapsed or refractory non-Hodgkin's lymphoma in an individualin need thereof, comprising: administering to the individual atherapeutically-effective amount of(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.In some embodiments, the non-Hodgkin's lymphoma is relapsed orrefractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractorymantle cell lymphoma, or relapsed or refractory follicular lymphoma.

Non-Hodgkin lymphomas (NHL) are a diverse group of malignancies that arepredominately of B-cell origin. NHL may develop in any organs associatedwith lymphatic system such as spleen, lymph nodes or tonsils and canoccur at any age. NHL is often marked by enlarged lymph nodes, fever,and weight loss. NHL is classified as either B-cell or T-cell NHL.Lymphomas related to lymphoproliferative disorders following bone marrowor stem cell transplantation are usually B-cell NHL. In the WorkingFormulation classification scheme, NHL has been divided into low-,intermediate-, and high-grade categories by virtue of their naturalhistories (see “The Non-Hodgkin's Lymphoma Pathologic ClassificationProject,” Cancer 49 (1982):2112-2135). The low-grade lymphomas areindolent, with a median survival of 5 to 10 years (Horning and Rosenberg(1984) N. Engl. J. Med. 311:1471-1475). Although chemotherapy can induceremissions in the majority of indolent lymphomas, cures are rare andmost patients eventually relapse, requiring further therapy. Theintermediate- and high-grade lymphomas are more aggressive tumors, butthey have a greater chance for cure with chemotherapy. However, asignificant proportion of these patients will relapse and requirefurther treatment.

A non-limiting list of the B-cell NHL includes Burkitt's lymphoma (e.g.,Endemic Burkitt's Lymphoma and Sporadic Burkitt's Lymphoma), CutaneousB-Cell Lymphoma, Cutaneous Marginal Zone Lymphoma (MZL), Diffuse LargeCell Lymphoma (DLBCL), Diffuse Mixed Small and Large Cell Lympoma,Diffuse Small Cleaved Cell, Diffuse Small Lymphocytic Lymphoma,Extranodal Marginal Zone B-cell lymphoma, follicular lymphoma,Follicular Small Cleaved Cell (Grade 1), Follicular Mixed Small Cleavedand Large Cell (Grade 2), Follicular Large Cell (Grade 3), IntravascularLarge B-Cell Lymphoma, Intravascular Lymphomatosis, Large CellImmunoblastic Lymphoma, Large Cell Lymphoma (LCL), LymphoblasticLymphoma, MALT Lymphoma, Mantle Cell Lymphoma (MCL), immunoblastic largecell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma,chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL),extranodal marginal zone B-cell lymphoma-mucosa-associated lymphoidtissue (MALT) lymphoma, Mediastinal Large B-Cell Lymphoma, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma,primary mediastinal B-cell lymphoma, lymphoplasmocytic lymphoma, hairycell leukemia, Waldenstrom's Macroglobulinemia, and primary centralnervous system (CNS) lymphoma. Additional non-Hodgkin's lymphomas arecontemplated within the scope of the present invention and apparent tothose of ordinary skill in the art.

DLBCL

Disclosed herein, in certain embodiments, is a method for treating aDLCBL in an individual in need thereof, comprising: (a) administering tothe individual an amount of an irreversible Btk inhibitor sufficient tomobilize a plurality of cells from the malignancy; and (b) analyzing themobilized plurality of cells. In some embodiments, the amount of theirreversible Btk inhibitor is sufficient to induce lymphocytosis of aplurality of cells from the malignancy. In some embodiments, analyzingthe mobilized plurality of cells comprises measuring the peripheralblood concentration of the mobilized plurality of cells. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells increases as compared to the concentrationbefore administration of the Btk inhibitor. In some embodiments,administering the second cancer treatment regimen occurs after asubsequent decrease in peripheral blood concentration of the mobilizedplurality of cells. In some embodiments, analyzing the mobilizedplurality of cells comprises measuring the duration of an increase inthe peripheral blood concentration of the mobilized plurality of cellsas compared to the concentration before administration of the Btkinhibitor. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells has increasedfor a predetermined length of time. In some embodiments, analyzing themobilized plurality of cells comprises counting the number of mobilizedplurality of cells in the peripheral blood. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time.

As used herein, the term “Diffuse large B-cell lymphoma (DLBCL)” refersto a neoplasm of the germinal center B lymphocytes with a diffuse growthpattern and a high-intermediate proliferation index. DLBCLs representapproximately 30% of all lymphomas and may present with severalmorphological variants including the centroblastic, immunoblastic,T-cell/histiocyte rich, anaplastic and plasmoblastic subtypes. Genetictests have shown that there are different subtypes of DLBCL. Thesesubtypes seem to have different outlooks (prognoses) and responses totreatment. DLBCL can affect any age group but occurs mostly in olderpeople (the average age is mid-60s).

Disclosed herein, in certain embodiments, is a method for treatingdiffuse large B-cell lymphoma, activated B cell-like subtype(ABC-DLBCL), in an individual in need thereof, comprising: administeringto the individual an irreversible Btk inhibitor in an amount from 300mg/day up to, and including, 1000 mg/day. The ABC subtype of diffuselarge B-cell lymphoma (ABC-DLBCL) is thought to arise from post germinalcenter B cells that are arrested during plasmatic differentiation. TheABC subtype of DLBCL (ABC-DLBCL) accounts for approximately 30% totalDLBCL diagnoses. It is considered the least curable of the DLBCLmolecular subtypes and, as such, patients diagnosed with the ABC-DLBCLtypically display significantly reduced survival rates compared withindividuals with other types of DLCBL. ABC-DLBCL is most commonlyassociated with chromosomal translocations deregulating the germinalcenter master regulator BCL6 and with mutations inactivating the PRDM1gene, which encodes a transcriptional repressor required for plasma celldifferentiation.

A particularly relevant signaling pathway in the pathogenesis ofABC-DLBCL is the one mediated by the nuclear factor (NF)-κBtranscription complex. The NF-κB family comprises 5 members (p50, p52,p65, c-rel and RelB) that form homo- and heterodimers and function astranscriptional factors to mediate a variety of proliferation,apoptosis, inflammatory and immune responses and are critical for normalB-cell development and survival. NF-κB is widely used by eukaryoticcells as a regulator of genes that control cell proliferation and cellsurvival. As such, many different types of human tumors havemisregulated NF-κB: that is, NF-κB is constitutively active. ActiveNF-κB turns on the expression of genes that keep the cell proliferatingand protect the cell from conditions that would otherwise cause it todie via apoptosis.

The dependence of ABC DLBCLs on NF-kB depends on a signaling pathwayupstream of IkB kinase comprised of CARD11, BCL10 and MALT1 (the CBMcomplex). Interference with the CBM pathway extinguishes NF-kB signalingin ABC DLBCL cells and induces apoptosis. The molecular basis forconstitutive activity of the NF-kB pathway is a subject of currentinvestigation but some somatic alterations to the genome of ABC DLBCLsclearly invoke this pathway. For example, somatic mutations of thecoiled-coil domain of CARD11 in DLBCL render this signaling scaffoldprotein able to spontaneously nucleate protein-protein interaction withMALT1 and BCL10, causing IKK activity and NF-kB activation. Constitutiveactivity of the B cell receptor signaling pathway has been implicated inthe activation of NF-kB in ABC DLBCLs with wild type CARD11, and this isassociated with mutations within the cytoplasmic tails of the B cellreceptor subunits CD79A and CD79B. Oncogenic activating mutations in thesignaling adapter MYD88 activate NF-kB and synergize with B cellreceptor signaling in sustaining the survival of ABC DLBCL cells. Inaddition, inactivating mutations in a negative regulator of the NF-kBpathway, A20, occur almost exclusively in ABC DLBCL.

Indeed, genetic alterations affecting multiple components of the NF-κBsignaling pathway have been recently identified in more than 50% ofABC-DLBCL patients, where these lesions promote constitutive NF-κBactivation, thereby contributing to lymphoma growth. These includemutations of CARD11 (˜10% of the cases), a lymphocyte-specificcytoplasmic scaffolding protein that—together with MALT1 and BCL10—formsthe BCR signalosome, which relays signals from antigen receptors to thedownstream mediators of NF-κB activation. An even larger fraction ofcases (˜30%) carry biallelic genetic lesions inactivating the negativeNF-κB regulator A20. Further, high levels of expression of NF-κB targetgenes have been observed in ABC-DLBCL tumor samples. See, e.g., U. Kleinet al., (2008), Nature Reviews Immunology 8:22-23; R. E. Davis et al.,(2001), Journal of Experimental Medicine 194:1861-1874; G. Lentz et al.,(2008), Science 319:1676-1679; M. Compagno et al., (2009), Nature459:712-721; and L. Srinivasan et al., (2009), Cell 139:573-586).

DLBCL cells of the ABC subtype, such as OCI-Ly10, have chronic activeBCR signalling and are very sensitive to the Btk inhibitors describedherein. The irreversible Btk inhibitors described herein potently andirreversibly inhibit the growth of OCI-Ly10 (EC50 continuous exposure=10nM, EC50 1 hour pulse=50 nM). In addition, induction of apoptosis, asshown by capsase activation, Annexin-V flow cytometry and increase insub-G0 fraction is observed in OCILy10. Both sensitive and resistantcells express Btk at similar levels, and the active site of Btk is fullyoccupied by the inhibitor in both as shown using a fluorescently labeledaffinity probe. OCI-Ly10 cells are shown to have chronically active BCRsignalling to NF-kB which is dose dependently inhibited by the Btkinhibitors described herein. The activity of Btk inhibitors in the celllines studied herein are also characterized by comparing signaltransduction profiles (Btk, PLCγ, ERK, NF-kB, AKT), cytokine secretionprofiles and mRNA expression profiles, both with and without BCRstimulation, and observed significant differences in these profiles thatlead to clinical biomarkers that identify the most sensitive patientpopulations to Btk inhibitor treatment. See U.S. Pat. No. 7,711,492 andStaudt et al., Nature, Vol. 463, Jan. 7, 2010, pp. 88-92, the contentsof which are incorporated by reference in their entirety.

Follicular Lymphoma

Disclosed herein, in certain embodiments, is a method for treating afollicular lymphoma in an individual in need thereof, comprising: (a)administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

As used herein, the term “follicular lymphoma” refers to any of severaltypes of non-Hodgkin's lymphoma in which the lymphomatous cells areclustered into nodules or follicles. The term follicular is used becausethe cells tend to grow in a circular, or nodular, pattern in lymphnodes. The average age for people with this lymphoma is about 60.

CLL/SLL

Disclosed herein, in certain embodiments, is a method for treating a CLLor SLL in an individual in need thereof, comprising: (a) administeringto the individual an amount of an irreversible Btk inhibitor sufficientto mobilize a plurality of cells from the malignancy; and (b) analyzingthe mobilized plurality of cells. In some embodiments, the CLL or SLL ishigh-risk. In some embodiments, the amount of the irreversible Btkinhibitor is sufficient to induce lymphocytosis of a plurality of cellsfrom the malignancy. In some embodiments, analyzing the mobilizedplurality of cells comprises measuring the peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the peripheral blood concentration of the mobilizedplurality of cells increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein peripheral blood concentration of the mobilized plurality of cells.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the peripheral bloodconcentration of the mobilized plurality of cells as compared to theconcentration before administration of the Btk inhibitor. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells has increased for a predetermined length oftime. In some embodiments, analyzing the mobilized plurality of cellscomprises counting the number of mobilized plurality of cells in theperipheral blood. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the number ofmobilized plurality of cells in the peripheral blood increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time.

Chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL)are commonly thought as the same disease with slightly differentmanifestations. Where the cancerous cells gather determines whether itis called CLL or SLL. When the cancer cells are primarily found in thelymph nodes, lima bean shaped structures of the lymphatic system (asystem primarily of tiny vessels found in the body), it is called SLL.SLL accounts for about 5% to 10% of all lymphomas. When most of thecancer cells are in the bloodstream and the bone marrow, it is calledCLL.

Both CLL and SLL are slow-growing diseases, although CLL, which is muchmore common, tends to grow slower. CLL and SLL are treated the same way.They are usually not considered curable with standard treatments, butdepending on the stage and growth rate of the disease, most patientslive longer than 10 years. Occasionally over time, these slow-growinglymphomas may transform into a more aggressive type of lymphoma.

Chronic lymphoid leukemia (CLL) is the most common type of leukemia. Itis estimated that 100,760 people in the United States are living with orare in remission from CLL. Most (>75%) people newly diagnosed with CLLare over the age of 50. Currently CLL treatment focuses on controllingthe disease and its symptoms rather than on an outright cure. CLL istreated by chemotherapy, radiation therapy, biological therapy, or bonemarrow transplantation. Symptoms are sometimes treated surgically(splenectomy removal of enlarged spleen) or by radiation therapy(“de-bulking” swollen lymph nodes). Though CLL progresses slowly in mostcases, it is considered generally incurable. Certain CLLs are classifiedas high-risk. As used herein, “high risk CLL” means CLL characterized byat least one of the following 1) 17p13-; 2) 11q22-; 3) unmutated IgVHtogether with ZAP-70+ and/or CD38+; or 4) trisomy 12.

CLL treatment is typically administered when the patient's clinicalsymptoms or blood counts indicate that the disease has progressed to apoint where it may affect the patient's quality of life.

Small lymphocytic leukemia (SLL) is very similar to CLL described supra,and is also a cancer of B-cells. In SLL the abnormal lymphocytes mainlyaffect the lymph nodes. However, in CLL the abnormal cells mainly affectthe blood and the bone marrow. The spleen may be affected in bothconditions. SLL accounts for about 1 in 25 of all cases of non-Hodgkinlymphoma. It can occur at any time from young adulthood to old age, butis rare under the age of 50. SLL is considered an indolent lymphoma.This means that the disease progresses very slowly, and patients tend tolive many years after diagnosis. However, most patients are diagnosedwith advanced disease, and although SLL responds well to a variety ofchemotherapy drugs, it is generally considered to be incurable. Althoughsome cancers tend to occur more often in one gender or the other, casesand deaths due to SLL are evenly split between men and women. Theaverage age at the time of diagnosis is 60 years.

Although SLL is indolent, it is persistently progressive. The usualpattern of this disease is one of high response rates to radiationtherapy and/or chemotherapy, with a period of disease remission. This isfollowed months or years later by an inevitable relapse. Re-treatmentleads to a response again, but again the disease will relapse. Thismeans that although the short-term prognosis of SLL is quite good, overtime, many patients develop fatal complications of recurrent disease.Considering the age of the individuals typically diagnosed with CLL andSLL, there is a need in the art for a simple and effective treatment ofthe disease with minimum side-effects that do not impede on thepatient's quality of life. The instant invention fulfills this longstanding need in the art.

Mantle Cell Lymphoma

Disclosed herein, in certain embodiments, is a method for treating aMantle cell lymphoma in an individual in need thereof, comprising: (a)administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

As used herein, the term, “Mantle cell lymphoma” refers to a subtype ofB-cell lymphoma, due to CD5 positive antigen-naive pregerminal centerB-cell within the mantle zone that surrounds normal germinal centerfollicles. MCL cells generally over-express cyclin D1 due to a t(11:14)chromosomal translocation in the DNA. More specifically, thetranslocation is at t(11;14)(q13;q32). Only about 5% of lymphomas are ofthis type. The cells are small to medium in size. Men are affected mostoften. The average age of patients is in the early 60s. The lymphoma isusually widespread when it is diagnosed, involving lymph nodes, bonemarrow, and, very often, the spleen. Mantle cell lymphoma is not a veryfast growing lymphoma, but is difficult to treat.

Marginal Zone B-Cell Lymphoma

Disclosed herein, in certain embodiments, is a method for treating amarginal zone B-cell lymphoma in an individual in need thereof,comprising: (a) administering to the individual an amount of anirreversible Btk inhibitor sufficient to mobilize a plurality of cellsfrom the malignancy; and (b) analyzing the mobilized plurality of cells.In some embodiments, the amount of the irreversible Btk inhibitor issufficient to induce lymphocytosis of a plurality of cells from themalignancy. In some embodiments, analyzing the mobilized plurality ofcells comprises measuring the peripheral blood concentration of themobilized plurality of cells. In some embodiments, the method furthercomprises administering a second cancer treatment regimen after theperipheral blood concentration of the mobilized plurality of cellsincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

As used herein, the term “marginal zone B-cell lymphoma” refers to agroup of related B-cell neoplasms that involve the lymphoid tissues inthe marginal zone, the patchy area outside the follicular mantle zone.Marginal zone lymphomas account for about 5% to 10% of lymphomas. Thecells in these lymphomas look small under the microscope. There are 3main types of marginal zone lymphomas including extranodal marginal zoneB-cell lymphomas, nodal marginal zone B-cell lymphoma, and splenicmarginal zone lymphoma.

MALT

Disclosed herein, in certain embodiments, is a method for treating aMALT in an individual in need thereof, comprising: (a) administering tothe individual an amount of an irreversible Btk inhibitor sufficient tomobilize a plurality of cells from the malignancy; and (b) analyzing themobilized plurality of cells. In some embodiments, the amount of theirreversible Btk inhibitor is sufficient to induce lymphocytosis of aplurality of cells from the malignancy. In some embodiments, analyzingthe mobilized plurality of cells comprises measuring the peripheralblood concentration of the mobilized plurality of cells. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells increases as compared to the concentrationbefore administration of the Btk inhibitor. In some embodiments,administering the second cancer treatment regimen occurs after asubsequent decrease in peripheral blood concentration of the mobilizedplurality of cells. In some embodiments, analyzing the mobilizedplurality of cells comprises measuring the duration of an increase inthe peripheral blood concentration of the mobilized plurality of cellsas compared to the concentration before administration of the Btkinhibitor. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells has increasedfor a predetermined length of time. In some embodiments, analyzing themobilized plurality of cells comprises counting the number of mobilizedplurality of cells in the peripheral blood. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time.

The term “mucosa-associated lymphoid tissue (MALT) lymphoma”, as usedherein, refers to extranodal manifestations of marginal-zone lymphomas.Most MALT lymphoma are a low grade, although a minority either manifestinitially as intermediate-grade non-Hodgkin lymphoma (NHL) or evolvefrom the low-grade form. Most of the MALT lymphoma occur in the stomach,and roughly 70% of gastric MALT lymphoma are associated withHelicobacter pylori infection. Several cytogenetic abnormalities havebeen identified, the most common being trisomy 3 or t(11;18). Many ofthese other MALT lymphoma have also been linked to infections withbacteria or viruses. The average age of patients with MALT lymphoma isabout 60.

Nodal Marginal Zone B-Cell Lymphoma

Disclosed herein, in certain embodiments, is a method for treating anodal marginal zone B-cell lymphoma in an individual in need thereof,comprising: (a) administering to the individual an amount of anirreversible Btk inhibitor sufficient to mobilize a plurality of cellsfrom the malignancy; and (b) analyzing the mobilized plurality of cells.In some embodiments, the amount of the irreversible Btk inhibitor issufficient to induce lymphocytosis of a plurality of cells from themalignancy. In some embodiments, analyzing the mobilized plurality ofcells comprises measuring the peripheral blood concentration of themobilized plurality of cells. In some embodiments, the method furthercomprises administering a second cancer treatment regimen after theperipheral blood concentration of the mobilized plurality of cellsincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

The term “nodal marginal zone B-cell lymphoma” refers to an indolentB-cell lymphoma that is found mostly in the lymph nodes. The disease israre and only accounts for 1% of all Non-Hodgkin's Lymphomas (NHL). Itis most commonly diagnosed in older patients, with women moresusceptible than men. The disease is classified as a marginal zonelymphoma because the mutation occurs in the marginal zone of theB-cells. Due to its confinement in the lymph nodes, this disease is alsoclassified as nodal.

Splenic Marginal Zone B-Cell Lymphoma

Disclosed herein, in certain embodiments, is a method for treating asplenic marginal zone B-cell lymphoma in an individual in need thereof,comprising: (a) administering to the individual an amount of anirreversible Btk inhibitor sufficient to mobilize a plurality of cellsfrom the malignancy; and (b) analyzing the mobilized plurality of cells.In some embodiments, the amount of the irreversible Btk inhibitor issufficient to induce lymphocytosis of a plurality of cells from themalignancy. In some embodiments, analyzing the mobilized plurality ofcells comprises measuring the peripheral blood concentration of themobilized plurality of cells. In some embodiments, the method furthercomprises administering a second cancer treatment regimen after theperipheral blood concentration of the mobilized plurality of cellsincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

The term “splenic marginal zone B-cell lymphoma” refers to specificlow-grade small B-cell lymphoma that is incorporated in the World HealthOrganization classification. Characteristic features are splenomegaly,moderate lymphocytosis with villous morphology, intrasinusoidal patternof involvement of various organs, especially bone marrow, and relativeindolent course. Tumor progression with increase of blastic forms andaggressive behavior are observed in a minority of patients. Molecularand cytogenetic studies have shown heterogeneous results probablybecause of the lack of standardized diagnostic criteria.

Burkitt Lymphoma

Disclosed herein, in certain embodiments, is a method for treating aBurkitt lymphoma in an individual in need thereof, comprising: (a)administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

The term “Burkitt lymphoma” refers to a type of Non-Hodgkin Lymphoma(NHL) that commonly affects children. It is a highly aggressive type ofB-cell lymphoma that often starts and involves body parts other thanlymph nodes. In spite of its fast-growing nature, Burkitt's lymphoma isoften curable with modern intensive therapies. There are two broad typesof Burkitt's lymphoma—the sporadic and the endemic varieties:

Endemic Burkitt's lymphoma: The disease involves children much more thanadults, and is related to Epstein Barr Virus (EBV) infection in 95%cases. It occurs primarily is equatorial Africa, where about half of allchildhood cancers are Burkitt's lymphoma. It characteristically has ahigh chance of involving the jawbone, a rather distinctive feature thatis rare in sporadic Burkitt's. It also commonly involves the abdomen.

Sporadic Burkitt's lymphoma: The type of Burkitt's lymphoma that affectsthe rest of the world, including Europe and the Americas is the sporadictype. Here too, it's mainly a disease in children. The link betweenEpstein Barr Virus (EBV) is not as strong as with the endemic variety,though direct evidence of EBV infection is present in one out of fivepatients. More than the involvement of lymph nodes, it is the abdomenthat is notably affected in more than 90% of the children. Bone marrowinvolvement is more common than in the sporadic variety.

Waldenstrom Macroglobulinemia

Disclosed herein, in certain embodiments, is a method for treating aWaldenstrom macroglobulinemia in an individual in need thereof,comprising: (a) administering to the individual an amount of anirreversible Btk inhibitor sufficient to mobilize a plurality of cellsfrom the malignancy; and (b) analyzing the mobilized plurality of cells.In some embodiments, the amount of the irreversible Btk inhibitor issufficient to induce lymphocytosis of a plurality of cells from themalignancy. In some embodiments, analyzing the mobilized plurality ofcells comprises measuring the peripheral blood concentration of themobilized plurality of cells. In some embodiments, the method furthercomprises administering a second cancer treatment regimen after theperipheral blood concentration of the mobilized plurality of cellsincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

The term “Waldenstrom macroglobulinemia”, also known aslymphoplasmacytic lymphoma, is cancer involving a subtype of white bloodcells called lymphocytes. It is characterized by an uncontrolled clonalproliferation of terminally differentiated B lymphocytes. It is alsocharacterized by the lymphoma cells making an antibody calledimmunoglobulin M (IgM). The IgM antibodies circulate in the blood inlarge amounts, and cause the liquid part of the blood to thicken, likesyrup. This can lead to decreased blood flow to many organs, which cancause problems with vision (because of poor circulation in blood vesselsin the back of the eyes) and neurological problems (such as headache,dizziness, and confusion) caused by poor blood flow within the brain.Other symptoms can include feeling tired and weak, and a tendency tobleed easily. The underlying etiology is not fully understood but anumber of risk factors have been identified, including the locus 6p21.3on chromosome 6. There is a 2- to 3-fold risk increase of developing WMin people with a personal history of autoimmune diseases withautoantibodies and particularly elevated risks associated withhepatitis, human immunodeficiency virus, and rickettsiosis.

Multiple Myeloma

Disclosed herein, in certain embodiments, is a method for treating amyeloma in an individual in need thereof, comprising: (a) administeringto the individual an amount of an irreversible Btk inhibitor sufficientto mobilize a plurality of cells from the malignancy; and (b) analyzingthe mobilized plurality of cells. In some embodiments, the amount of theirreversible Btk inhibitor is sufficient to induce lymphocytosis of aplurality of cells from the malignancy. In some embodiments, analyzingthe mobilized plurality of cells comprises measuring the peripheralblood concentration of the mobilized plurality of cells. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells increases as compared to the concentrationbefore administration of the Btk inhibitor. In some embodiments,administering the second cancer treatment regimen occurs after asubsequent decrease in peripheral blood concentration of the mobilizedplurality of cells. In some embodiments, analyzing the mobilizedplurality of cells comprises measuring the duration of an increase inthe peripheral blood concentration of the mobilized plurality of cellsas compared to the concentration before administration of the Btkinhibitor. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells has increasedfor a predetermined length of time. In some embodiments, analyzing themobilized plurality of cells comprises counting the number of mobilizedplurality of cells in the peripheral blood. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time.

Disclosed herein, in certain embodiments, is a method for treating amultiple myeloma in an individual in need thereof, comprising: (a)administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

Multiple myeloma, also known as MM, myeloma, plasma cell myeloma, or asKahler's disease (after Otto Kahler) is a cancer of the white bloodcells known as plasma cells. A type of B cell, plasma cells are acrucial part of the immune system responsible for the production ofantibodies in humans and other vertebrates. They are produced in thebone marrow and are transported through the lymphatic system.

Leukemia

Disclosed herein, in certain embodiments, is a method for treating aleukemia in an individual in need thereof, comprising: (a) administeringto the individual an amount of an irreversible Btk inhibitor sufficientto mobilize a plurality of cells from the malignancy; and (b) analyzingthe mobilized plurality of cells. In some embodiments, the amount of theirreversible Btk inhibitor is sufficient to induce lymphocytosis of aplurality of cells from the malignancy. In some embodiments, analyzingthe mobilized plurality of cells comprises measuring the peripheralblood concentration of the mobilized plurality of cells. In someembodiments, the method further comprises administering a second cancertreatment regimen after the peripheral blood concentration of themobilized plurality of cells increases as compared to the concentrationbefore administration of the Btk inhibitor. In some embodiments,administering the second cancer treatment regimen occurs after asubsequent decrease in peripheral blood concentration of the mobilizedplurality of cells. In some embodiments, analyzing the mobilizedplurality of cells comprises measuring the duration of an increase inthe peripheral blood concentration of the mobilized plurality of cellsas compared to the concentration before administration of the Btkinhibitor. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells has increasedfor a predetermined length of time. In some embodiments, analyzing themobilized plurality of cells comprises counting the number of mobilizedplurality of cells in the peripheral blood. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodincreases as compared to the concentration before administration of theBtk inhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in the number ofmobilized plurality of cells in the peripheral blood. In someembodiments, analyzing the mobilized plurality of cells comprisesmeasuring the duration of an increase in the number of mobilizedplurality of cells in the peripheral blood as compared to the numberbefore administration of the Btk inhibitor. In some embodiments, themethod further comprises administering a second cancer treatment regimenafter the number of mobilized plurality of cells in the peripheral bloodhas increased for a predetermined length of time.

Leukemia is a cancer of the blood or bone marrow characterized by anabnormal increase of blood cells, usually leukocytes (white bloodcells). Leukemia is a broad term covering a spectrum of diseases. Thefirst division is between its acute and chronic forms: (i) acuteleukemia is characterized by the rapid increase of immature blood cells.This crowding makes the bone marrow unable to produce healthy bloodcells. Immediate treatment is required in acute leukemia due to therapid progression and accumulation of the malignant cells, which thenspill over into the bloodstream and spread to other organs of the body.Acute forms of leukemia are the most common forms of leukemia inchildren; (ii) chronic leukemia is distinguished by the excessive buildup of relatively mature, but still abnormal, white blood cells.Typically taking months or years to progress, the cells are produced ata much higher rate than normal cells, resulting in many abnormal whiteblood cells in the blood. Chronic leukemia mostly occurs in olderpeople, but can theoretically occur in any age group. Additionally, thediseases are subdivided according to which kind of blood cell isaffected. This split divides leukemias into lymphoblastic or lymphocyticleukemias and myeloid or myelogenous leukemias: (i) lymphoblastic orlymphocytic leukemias, the cancerous change takes place in a type ofmarrow cell that normally goes on to form lymphocytes, which areinfection-fighting immune system cells; (ii) myeloid or myelogenousleukemias, the cancerous change takes place in a type of marrow cellthat normally goes on to form red blood cells, some other types of whitecells, and platelets.

Within these main categories, there are several subcategories including,but not limited to, Acute lymphoblastic leukemia (ALL), Acutemyelogenous leukemia (AML), Chronic myelogenous leukemia (CML), andHairy cell leukemia (HCL).

Btk Inhibitors

Also presented herein are methods for treating a cancer such as by wayof example only, a BCLD, in a subject wherein the subject has beentreated with a dosing regimen of a Btk inhibitor. In the followingdescription of irreversible Btk compounds suitable for use in themethods described herein, definitions of referred-to standard chemistryterms may be found in reference works (if not otherwise defined herein),including Carey and Sundberg “Advanced Organic Chemistry 4th Ed.” Vols.A (2000) and B (2001), Plenum Press, New York. Unless otherwiseindicated, conventional methods of mass spectroscopy, NMR, HPLC, proteinchemistry, biochemistry, recombinant DNA techniques and pharmacology,within the ordinary skill of the art are employed. In addition, nucleicacid and amino acid sequences for Btk (e.g., human Btk) are known in theart as disclosed in, e.g., U.S. Pat. No. 6,326,469. Unless specificdefinitions are provided, the nomenclature employed in connection with,and the laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those known in the art. Standard techniques can beused for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

The Btk inhibitor compounds described herein are selective for Btk andkinases having a cysteine residue in an amino acid sequence position ofthe tyrosine kinase that is homologous to the amino acid sequenceposition of cysteine 481 in Btk. Generally, an irreversible inhibitorcompound of Btk used in the methods described herein is identified orcharacterized in an in vitro assay, e.g., an acellular biochemical assayor a cellular functional assay. Such assays are useful to determine anin vitro IC₅₀ for an irreversible Btk inhibitor compound.

For example, an acellular kinase assay can be used to determine Btkactivity after incubation of the kinase in the absence or presence of arange of concentrations of a candidate irreversible Btk inhibitorcompound. If the candidate compound is in fact an irreversible Btkinhibitor, Btk kinase activity will not be recovered by repeat washingwith inhibitor-free medium. See, e.g., J. B. Smaill, et al. (1999), J.Med. Chem. 42(10):1803-1815. Further, covalent complex formation betweenBtk and a candidate irreversible Btk inhibitor is a useful indicator ofirreversible inhibition of Btk that can be readily determined by anumber of methods known in the art (e.g., mass spectrometry). Forexample, some irreversible Btk-inhibitor compounds can form a covalentbond with Cys 481 of Btk (e.g., via a Michael reaction).

Cellular functional assays for Btk inhibition include measuring one ormore cellular endpoints in response to stimulating a Btk-mediatedpathway in a cell line (e.g., BCR activation in Ramos cells) in theabsence or presence of a range of concentrations of a candidateirreversible Btk inhibitor compound. Useful endpoints for determining aresponse to BCR activation include, e.g., autophosphorylation of Btk,phosphorylation of a Btk target protein (e.g., PLC-γ), and cytoplasmiccalcium flux.

High throughput assays for many acellular biochemical assays (e.g.,kinase assays) and cellular functional assays (e.g., calcium flux) arewell known to those of ordinary skill in the art. In addition, highthroughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. Automated systems thereby allow theidentification and characterization of a large number of irreversibleBtk compounds without undue effort.

In some embodiments, the Btk inhibitor is selected from the groupconsisting of a small organic molecule, a macromolecule, a peptide or anon-peptide.

In some embodiments, the Btk inhibitor provided herein is a reversibleor irreversible inhibitor. In certain embodiments, the Btk inhibitor isan irreversible inhibitor.

In some embodiments, the irreversible Btk inhibitor forms a covalentbond with a cysteine sidechain of a Bruton's tyrosine kinase, a Bruton'styrosine kinase homolog, or a Btk tyrosine kinase cysteine homolog.

Irreversible Btk inhibitor compounds can use for the manufacture of amedicament for treating any of the foregoing conditions (e.g.,autoimmune diseases, inflammatory diseases, allergy disorders, B-cellproliferative disorders, or thromboembolic disorders).

In some embodiments, the irreversible Btk inhibitor compound used forthe methods described herein inhibits Btk or a Btk homolog kinaseactivity with an in vitro IC₅₀ of less than 10 μM. (e.g., less than 1μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.1,less than 0.08 μM, less than 0.06 μM, less than 0.05 μM, less than 0.04μM, less than 0.03 μM, less than less than 0.02 μM, less than 0.01, lessthan 0.008 μM, less than 0.006 μM, less than 0.005 μM, less than 0.004μM, less than 0.003 μM, less than less than 0.002 μM, less than 0.001,less than 0.00099 μM, less than 0.00098 μM, less than 0.00097 μM, lessthan 0.00096 μM, less than 0.00095 μM, less than 0.00094 μM, less than0.00093 μM, less than 0.00092, or less than 0.00090 μM).

In one embodiment, the irreversible Btk inhibitor compound selectivelyand irreversibly inhibits an activated form of its target tyrosinekinase (e.g., a phosphorylated form of the tyrosine kinase). Forexample, activated Btk is transphosphorylated at tyrosine 551. Thus, inthese embodiments the irreversible Btk inhibitor inhibits the targetkinase in cells only once the target kinase is activated by thesignaling events.

In other embodiments, the Btk inhibitor used in the methods describeherein has the structure of any of Formula (A), Formula (B), Formula(C), Formula (D), Formula (E), or Formula (F). Also described herein arepharmaceutically acceptable salts, pharmaceutically acceptable solvates,pharmaceutically active metabolites, and pharmaceutically acceptableprodrugs of such compounds. Pharmaceutical compositions that include atleast one such compound or a pharmaceutically acceptable salt,pharmaceutically acceptable solvate, pharmaceutically active metaboliteor pharmaceutically acceptable prodrug of such compound, are provided.In some embodiments, when compounds disclosed herein contain anoxidizable nitrogen atom, the nitrogen atom can be converted to anN-oxide by methods well known in the art. In certain embodiments,isomers and chemically protected forms of compounds having a structurerepresented by any of Formula (A), Formula (B), Formula (C), Formula(D), Formula (E), or Formula (F), are also provided.

Formula (A) is as follows:

wherein:

-   -   A is independently selected from N or CR₅;    -   R₁ is H, L₂-(substituted or unsubstituted alkyl),        L₂-(substituted or unsubstituted cycloalkyl), L₂-(substituted or        unsubstituted alkenyl), L₂-(substituted or unsubstituted        cycloalkenyl), L₂-(substituted or unsubstituted heterocycle),        L₂-(substituted or unsubstituted heteroaryl), or L₂-(substituted        or unsubstituted aryl), where L₂ is a bond, O, S, —S(═O),        —S(═O)₂, C(═O), -(substituted or unsubstituted C₁-C₆ alkyl), or        -(substituted or unsubstituted C₂-C₆ alkenyl);    -   R₂ and R₃ are independently selected from H, lower alkyl and        substituted lower alkyl;    -   R₄ is L₃-X-L₄-G, wherein,        -   L₃ is optional, and when present is a bond, optionally            substituted or unsubstituted alkyl, optionally substituted            or unsubstituted cycloalkyl, optionally substituted or            unsubstituted alkenyl, optionally substituted or            unsubstituted alkynyl;        -   X is optional, and when present is a bond, O, —C(═O), S,            —S(═O), —S(═O)₂, —NH, —NR₉, —NHC(O), —C(O)NH, —NR₉C(O),            —C(O)NR₉, —S(═O)₂NH, —NHS(═O)₂, —S(═O)₂NR₉—, —NR₉S(═O)₂,            —OC(O)NH—, —NHC(O)O—, —OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—,            —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl, aryl,            —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—,            —OC(═NR₁₁)—, or —C(═NR₁₁)O—;        -   L₄ is optional, and when present is a bond, substituted or            unsubstituted alkyl, substituted or unsubstituted            cycloalkyl, substituted or unsubstituted alkenyl,            substituted or unsubstituted alkynyl, substituted or            unsubstituted aryl, substituted or unsubstituted heteroaryl,            substituted or unsubstituted heterocycle;        -   or L₃, X and L₄ taken together form a nitrogen containing            heterocyclic ring;        -   G is

-   -    wherein,        -   R₆, R₇ and R₈ are independently selected from among H, lower            alkyl or substituted lower alkyl, lower heteroalkyl or            substituted lower heteroalkyl, substituted or unsubstituted            lower cycloalkyl, and substituted or unsubstituted lower            heterocycloalkyl;    -   R₅ is H, halogen, -L₆-(substituted or unsubstituted C₁-C₃        alkyl), -L₆-(substituted or unsubstituted C₂-C₄ alkenyl),        -L₆-(substituted or unsubstituted heteroaryl), or        -L₆-(substituted or unsubstituted aryl), wherein L₆ is a bond,        O, S, —S(═O), S(═O)₂, NH, C(O), —NHC(O)O, —OC(O)NH, —NHC(O), or        —C(O)NH;    -   each R₉ is independently selected from among H, substituted or        unsubstituted lower alkyl, and substituted or unsubstituted        lower cycloalkyl;    -   each R₁₀ is independently H, substituted or unsubstituted lower        alkyl, or substituted or unsubstituted lower cycloalkyl; or    -   two R₁₀ groups can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or    -   R₉ and R₁₀ can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or    -   each R₁₁ is independently selected from H, —S(═O)₂R₈,        —S(═O)₂NH₂, —C(O)R₈, —CN, —NO₂, heteroaryl, or heteroalkyl; and    -   pharmaceutically active metabolites, pharmaceutically acceptable        solvates, pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In one aspect are compounds having the structure of Formula (A1):

wherein

-   -   A is independently selected from N or CR₅;    -   R₁ is H, L₂-(substituted or unsubstituted alkyl),        L₂-(substituted or unsubstituted cycloalkyl), L₂-(substituted or        unsubstituted alkenyl), L₂-(substituted or unsubstituted        cycloalkenyl), L₂-(substituted or unsubstituted heterocycle),        L₂-(substituted or unsubstituted heteroaryl), or L₂-(substituted        or unsubstituted aryl), where L₂ is a bond, O, S, —S(═O),        —S(═O)₂, C(═O), -(substituted or unsubstituted C₁-C₆ alkyl), or        -(substituted or unsubstituted C₂-C₆ alkenyl);    -   R₂ and R₃ are independently selected from H, lower alkyl and        substituted lower alkyl;    -   R₄ is L₃-X-L₄-G, wherein,        -   L₃ is optional, and when present is a bond, or an optionally            substituted group selected from alkyl, heteroalkyl, aryl,            heteroaryl, alkylaryl, alkylheteroaryl, or            alkylheterocycloalkyl;        -   X is optional, and when present is a bond, O, —C(═O), S,            —S(═O), —S(═O)₂, —NH, —NR₉, —NHC(O), —C(O)NH, —NR₉C(O),            —C(O)NR₉, —S(═O)₂NH, —NHS(═O)₂, —S(═O)₂NR₉—, —NR₉S(═O)₂,            —OC(O)NH—, —NHC(O)O—, —OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—,            —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl, aryl,            —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)O—, —C(═NR₁₁)NR₁₀—,            —OC(═NR₁₁)—, or —C(═NR₁₁)O—;        -   L₄ is optional, and when present is a bond, substituted or            unsubstituted alkyl, substituted or unsubstituted            cycloalkyl, substituted or unsubstituted alkenyl,            substituted or unsubstituted alkynyl, substituted or            unsubstituted aryl, substituted or unsubstituted heteroaryl,            substituted or unsubstituted heterocycle;        -   or L₃, X and L₄ taken together form a nitrogen containing            heterocyclic ring, or an optionally substituted group            selected from alkyl, heteroalkyl, aryl, heteroaryl,            alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl;            -   G is

-   -    where R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either        -   R₇ and R₈ are H;            -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,                substituted or unsubstituted C₁-C₄heteroalkyl,                C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted                C₃-C₆cycloalkyl, substituted or unsubstituted                C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted                aryl, substituted or unsubstituted                C₂-C₈heterocycloalkyl, substituted or unsubstituted                heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),                C₁-C₈alkylethers, C₁-C₈alkylamides, or                C₁-C₄alkyl(C₂-C₈heterocycloalkyl);        -   R₆ and R₈ are H;            -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,                substituted or unsubstituted C₁-C₄heteroalkyl,                C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted                C₃-C₆cycloalkyl, substituted or unsubstituted                C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted                aryl, substituted or unsubstituted                C₂-C₈heterocycloalkyl, substituted or unsubstituted                heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),                C₁-C₈alkylethers, C₁-C₈alkylamides, or                C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or            -   R₆ and R₈ taken together form a bond;                -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,                    substituted or unsubstituted C₁-C₄heteroalkyl,                    C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                    C₁-C₈alkoxyalkylaminoalkyl, substituted or                    unsubstituted C₃-C₆cycloalkyl, substituted or                    unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted                    or unsubstituted aryl, substituted or unsubstituted                    C₂-C₈heterocycloalkyl, substituted or unsubstituted                    heteroaryl, C₁-C₄alkyl(aryl),                    C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,                    C₁-C₈alkylamides, or                    C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₅ is H, halogen, -L₆-(substituted or unsubstituted C₁-C₃        alkyl), -L₆-(substituted or unsubstituted C₂-C₄ alkenyl),        -L₆-(substituted or unsubstituted heteroaryl), or        -L₆-(substituted or unsubstituted aryl), wherein L₆ is a bond,        O, S, —S(═O), S(═O)₂, NH, C(O), —NHC(O)O, —OC(O)NH, —NHC(O), or        —C(O)NH;    -   each R₉ is independently selected from among H, substituted or        unsubstituted lower alkyl, and substituted or unsubstituted        lower cycloalkyl;    -   each R₁₀ is independently H, substituted or unsubstituted lower        alkyl, or substituted or unsubstituted lower cycloalkyl; or    -   two R₁₀ groups can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or    -   R₉ and R₁₀ can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or    -   each R₁₁ is independently selected from H, —S(═O)₂R₈,        —S(═O)₂NH₂, —C(O)R₈, —CN, —NO₂, heteroaryl, or heteroalkyl; and        pharmaceutically active metabolites, pharmaceutically acceptable        solvates, pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (A1). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (A1), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (A1). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (A1). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further embodiment, the compound of Formula (A) has the followingstructure of Formula (B):

wherein:

-   -   Y is alkyl or substituted alkyl, or a 4-, 5-, or 6-membered        cycloalkyl ring;    -   each R_(a) is independently H, halogen, —CF₃, —CN, —NO₂, OH,        NH₂, -L_(a)-(substituted or unsubstituted alkyl),        -L_(a)-(substituted or unsubstituted alkenyl),        -L_(a)-(substituted or unsubstituted heteroaryl), or        -L_(a)-(substituted or unsubstituted aryl), wherein L_(a) is a        bond, O, S, —S(═O), —S(═O)₂, NH, C(O), CH₂, —NHC(O)O, —NHC(O),        or —C(O)NH;    -   G is

wherein,

-   -   R₆, R₇ and R₈ are independently selected from among H, lower        alkyl or substituted lower alkyl, lower heteroalkyl or        substituted lower heteroalkyl, substituted or unsubstituted        lower cycloalkyl, and substituted or unsubstituted lower        heterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and    -   pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In further embodiments, G is selected from among

In further embodiments,

is selected from among

In a further embodiment, the compound of Formula (A1) has the followingstructure of Formula (B1):

wherein:

-   -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, and        alkyleneheterocycloalkylene;    -   each R_(a) is independently H, halogen, —CF₃, —CN, —NO₂, OH,        NH₂, -L_(a)-(substituted or unsubstituted alkyl),        -L_(a)-(substituted or unsubstituted alkenyl),        -L_(a)-(substituted or unsubstituted heteroaryl), or        -L_(a)-(substituted or unsubstituted aryl), wherein L_(a) is a        bond, O, S, —S(═O), —S(═O)₂, NH, C(O), CH₂, —NHC(O)O, —NHC(O),        or —C(O)NH;    -   G is

where R^(a) is H, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl; and either

-   -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₆ and R₈ taken together form a bond;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and    -   pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In further embodiments, G is selected from among

where R is H, alkyl, alkylhydroxy, heterocycloalkyl, heteroaryl,alkylalkoxy, alkylalkoxyalkyl.

In further embodiments,

is selected from among

In a further embodiment, the compound of Formula (B) has the followingstructure of Formula (C):

-   -   Y is alkyl or substituted alkyl, or a 4-, 5-, or 6-membered        cycloalkyl ring;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

wherein,

-   -   R₆, R₇ and R₈ are independently selected from among H, lower        alkyl or substituted lower alkyl, lower heteroalkyl or        substituted lower heteroalkyl, substituted or unsubstituted        lower cycloalkyl, and substituted or unsubstituted lower        heterocycloalkyl; and    -   pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In further embodiment, the compound of Formula (B1) has the followingstructure of Formula (C1):

-   -   Y is an optionally substituted group selected from among alkyl,        heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, and        alkylheterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

where R^(a) is H, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl; and either

-   -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₆ and R₈ taken together form a bond;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and    -   pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In a further or alternative embodiment, the “G” group of any of Formula(A1), Formula (B1), or Formula (C1) is any group that is used to tailorthe physical and biological properties of the molecule. Suchtailoring/modifications are achieved using groups which modulate Michaelacceptor chemical reactivity, acidity, basicity, lipophilicity,solubility and other physical properties of the molecule. The physicaland biological properties modulated by such modifications to G include,by way of example only, enhancing chemical reactivity of Michaelacceptor group, solubility, in vivo absorption, and in vivo metabolism.In addition, in vivo metabolism includes, by way of example only,controlling in vivo PK properties, off-target activities, potentialtoxicities associated with cypP450 interactions, drug-drug interactions,and the like. Further, modifications to G allow for the tailoring of thein vivo efficacy of the compound through the modulation of, by way ofexample, specific and non-specific protein binding to plasma proteinsand lipids and tissue distribution in vivo.

In another embodiment, provided herein is a compound of Formula (D).Formula (D) is as follows:

wherein:

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is a substituted or unsubstituted aryl, or a substituted or        unsubstituted heteroaryl;    -   Y is an optionally substituted group selected from among alkyl,        heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;    -   Z is C(═O), OC(═O), NHC(═O), C(═S), S(═O)_(x), OS(═O)_(x),        NHS(═O)_(x), where x is 1 or 2;    -   R₆, R₇, and R₈ are each independently selected from among H,        substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, substituted or unsubstituted        C₃-C₆cycloalkyl, substituted or unsubstituted        C₂-C₆heterocycloalkyl, C₁-C₆alkoxyalkyl, C₁-C₈alkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted C₁-C₄alkyl(aryl), substituted or        unsubstituted C₁-C₄alkyl(heteroaryl), substituted or        unsubstituted C₁-C₄alkyl(C₃-C₈cycloalkyl), or substituted or        unsubstituted C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond; and pharmaceutically        active metabolites, or pharmaceutically acceptable solvates,        pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In one embodiment are compounds having the structure of Formula (D1):

wherein

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is an optionally substituted aromatic carbocycle or an        aromatic heterocycle;    -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, and        alkyleneheterocycloalkylene, or combination thereof;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₆ and R₈ taken together form a bond;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   or combinations thereof; and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (D1). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (D1), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (D1). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (D1). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further embodiment, L_(a) is O.

In a further embodiment, Ar is phenyl.

In a further embodiment, Z is C(═O), NHC(═O), or NCH₃C(═O).

In a further embodiment, each of R₁, R₂, and R₃ is H.

In one embodiment is a compound of Formula (D1) wherein R₆, R₇, and R₈are all H. In another embodiment, R₆, R₇, and R₈ are not all H.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, L_(a) is CH₂, O, or NH. In other embodiments, L_(a) is O orNH. In yet other embodiments, L_(a) is O.

In some embodiments, Ar is a substituted or unsubstituted aryl. In yetother embodiments, Ar is a 6-membered aryl. In some other embodiments,Ar is phenyl.

In some embodiments, x is 2. In yet other embodiments, Z is C(═O),OC(═O), NHC(═O), S(═O)_(x), OS(═O)_(x), or NHS(═O)_(x). In some otherembodiments, Z is C(═O), NHC(═O), or S(═O)₂.

In some embodiments, R₇ and R₈ are independently selected from among H,unsubstituted C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstitutedC₁-C₄heteroalkyl, and substituted C₁-C₄heteroalkyl; or R₇ and R₈ takentogether form a bond. In yet other embodiments, each of R₇ and R₈ is H;or R₇ and R₈ taken together form a bond.

In some embodiments, R₆ is H, substituted or unsubstituted C₁-C₄alkyl,substituted or unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,C₁-C₂alkyl-N(C₁-C₃alkyl)₂, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), orC₁-C₄alkyl(C₂-C₈heterocycloalkyl). In some other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₆ alkoxyalkyl, C₁-C₂alkyl-N(C₁-C₃alkyl)₂,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl),or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In yet other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl),—CH₂—N(C₁-C₃alkyl)₂, C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-memberedheteroaryl). In some embodiments, R₆ is H, substituted or unsubstitutedC₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl), —CH₂—N(C₁-C₃alkyl)₂,C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-membered heteroaryl containing1 or 2 N atoms), or C₁-C₄alkyl(5- or 6-membered heterocycloalkylcontaining 1 or 2 N atoms).

In some embodiments, Y is an optionally substituted group selected fromamong alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. In otherembodiments, Y is an optionally substituted group selected from amongC₁-C₆alkyl, C₁-C₆heteroalkyl, 4-, 5-, 6- or 7-membered cycloalkyl, and4-, 5-, 6- or 7-membered heterocycloalkyl. In yet other embodiments, Yis an optionally substituted group selected from among C₁-C₆alkyl,C₁-C₆heteroalkyl, 5-, or 6-membered cycloalkyl, and 5-, or 6-memberedheterocycloalkyl containing 1 or 2 N atoms. In some other embodiments, Yis a 5-, or 6-membered cycloalkyl, or a 5-, or 6-memberedheterocycloalkyl containing 1 or 2 N atoms.

Any combination of the groups described above for the various variablesis contemplated herein. It is understood that substituents andsubstitution patterns on the compounds provided herein can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be synthesized by techniques known in theart, as well as those set forth herein.

In one embodiment the irreversible inhibitor of a kinase has thestructure of Formula (E):

wherein:

-   -   wherein

is a moiety that binds to the active site of a kinase, including atyrosine kinase, further including a Btk kinase cysteine homolog;

-   -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        heterocycloalkylene, cycloalkylene, alkylenearylene,        alkyleneheteroarylene, alkylenecycloalkylene, and        alkyleneheterocycloalkylene;    -   Z is C(═O), OC(═O), NHC(═O), NCH₃C(═O), C(═S), S(═O)_(x),        OS(═O)_(x), NHS(═O)_(x), where x is 1 or 2;    -   R₆, R₇, and R₈ are each independently selected from among H,        substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, substituted or unsubstituted        C₃-C₆cycloalkyl, substituted or unsubstituted        C₂-C₆heterocycloalkyl, C₁-C₆alkoxyalkyl, C₁-C₈alkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted C₁-C₄alkyl(aryl), substituted or        unsubstituted C₁-C₄alkyl(heteroaryl), substituted or        unsubstituted C₁-C₄alkyl(C₃-C₈cycloalkyl), or substituted or        unsubstituted C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond; and pharmaceutically        active metabolites, or pharmaceutically acceptable solvates,        pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In some embodiments,

is a substituted fused biaryl moiety selected from

In one aspect, provided herein are compounds of Formula (F). Formula (F)is as follows:

wherein

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is a substituted or unsubstituted aryl, or a substituted or        unsubstituted heteroaryl; and either    -   (a) Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene        and alkyleneheterocycloalkylene;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either

(i) R₆, R₇, and R₈ are each independently selected from among H,substituted or unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, substituted or unsubstituted C₃-C₆cycloalkyl,substituted or unsubstituted C₂-C₆heterocycloalkyl, C₁-C₆alkoxyalkyl,C₁-C₈alkylaminoalkyl, substituted or unsubstituted C₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted C₁-C₄alkyl(aryl), substitutedor unsubstituted C₁-C₄alkyl(heteroaryl), substituted or unsubstitutedC₁-C₄alkyl(C₃-C₈cycloalkyl), or substituted or unsubstitutedC₁-C₄alkyl(C₂-C₈heterocycloalkyl);

(ii) R₆ and R₈ are H;

-   -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl, C₁-C₈        hydroxyalkylaminoalkyl, C₁-C₈ alkoxyalkylaminoalkyl, substituted        or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted        C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted C₂-C₈heterocycloalkyl, substituted        or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),        C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides, or        C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or

(iii) R₇ and R₈ taken together form a bond;

-   -   R₆ is selected from among H, substituted or unsubstituted        C₁-C₄alkyl, substituted or unsubstituted C₁-C₄heteroalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₂-C₆heterocycloalkyl, C₁-C₆alkoxyalkyl,        C₁-C₈alkylaminoalkyl, substituted or unsubstituted        C₃-C₆cycloalkyl, substituted or unsubstituted aryl, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        C₁-C₄alkyl(aryl), substituted or unsubstituted        C₁-C₄alkyl(heteroaryl), substituted or unsubstituted        C₁-C₄alkyl(C₃-C₈cycloalkyl), or substituted or unsubstituted        C₁-C₄alkyl(C₂-C₈heterocycloalkyl) or    -   (b) Y is an optionally substituted group selected from        cycloalkylene or heterocycloalkylene;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either

(i) R₇ and R₈ are H;

-   -   R₆ is substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl, C₁-C₈        hydroxyalkylaminoalkyl, C₁-C₈ alkoxyalkylaminoalkyl, substituted        or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted        C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted C₂-C₈heterocycloalkyl, substituted        or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),        C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides, or        C₁-C₄alkyl(C₂-C₈heterocycloalkyl);

(ii) R₆ and R₈ are H;

-   -   R₇ is substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl, C₁-C₈        hydroxyalkylaminoalkyl, C₁-C₈ alkoxyalkylaminoalkyl, substituted        or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted        C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted aryl,        substituted or unsubstituted C₂-C₈heterocycloalkyl, substituted        or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),        C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides, or        C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or

(iii) R₇ and R₈ taken together form a bond;

-   -   R₆ is substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

Further embodiments of compounds of Formula (A), Formula (B), Formula(C), Formula (D), include, but are not limited to, compounds selectedfrom the group consisting of:

In still another embodiment, compounds provided herein are selected fromamong:

In one aspect, provided herein is a compound selected from among:1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Compound 4);(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-en-1-one(Compound 5);1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)sulfonylethene(Compound 6);1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-yn-1-one(Compound 8);1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Compound 9);N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)acrylamide(Compound 10);1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 11);1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 12);1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Compound 13);1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Compound 14); and(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 15).

In some embodiments, the Btk inhibitor is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.

In one embodiment, the Btk inhibitor isα-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13),AVL-101,4-tert-butyl-N-(3-(8-(phenylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)benzamide,5-(3-amino-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenylamino)pyrazin-2(1H)-one,N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)acetamide,4-tert-butyl-N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide,5-(3-(4-tert-butylbenzylamino)-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenylamino)pyrazin-2(1H)-one,5-(3-(3-tert-butylbenzylamino)-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenylamino)pyrazin-2(1H)-one,3-tert-butyl-N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide,6-tert-butyl-N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)nicotinamide,and terreic acid.

Throughout the specification, groups and substituents thereof can bechosen by one skilled in the field to provide stable moieties andcompounds.

In certain embodiments, any of the Btk inhibitors and/or the secondagent provided herein for the invention methods is included in apharmaceutical composition comprising: i) a physiologically acceptablecarrier, diluent, and/or excipient.

In some embodiments, the Btk inhibitor of the invention methods isadministered at a dose of from about 1.25 mg/kg/day to about 12.5mg/kg/day. In certain embodiments, the Btk inhibitor is administered ata dose selected from the group consisting of about 1.25 mg/kg/day, about2.5 mg/kg/day, about 5 mg/kg/day, about 8.3 mg/kg/day, or about 12.5mg/kg/day.

In some embodiments provide the biomarkers in accordance with thepractice of the present invention is selected from ZAP-70, CD5,t(14;18), CD38, β-2 microglobulin, p53 mutational status, ATM mutationalstatus, chromosome 17p deletion, chromosome 11q deletion, surface orcytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22,CD11c, CD 103, chromosome 7q deletion, and V_(H) mutational status.

In some embodiments, determining the expression or presence of one ormore biomarkers from one or more subpopulation of lymphocytes is of acombination of biomarkers. In certain embodiments, the combination ofbiomarkers is CD19 and CD5 or CD20 and CD5.

In other embodiments, the second agent is administered at a dose of fromabout 1.25 mg/kg/day to about 12.5 mg/kg/day. In certain embodiments,the second agent is administered at a dose selected from the groupconsisting of about 1.25 mg/kg/day, about 2.5 mg/kg/day, about 5mg/kg/day, about 8.3 mg/kg/day, or about 12.5 mg/kg/day. The dosage ofthe second agent is based on the determined expression or presence ofone or more biomarkers from one or more subpopulation of lymphocytes. Aperson skilled in the art such as a physician can readily determine thesuitable regimen (e.g. dosage of the second agent) based on thediagnostic results.

In other embodiments, the present invention provides methods fortreating a cancer comprising determining the expression or presence ofone or more biomarkers from one or more subpopulation of lymphocytes ina subject that has received a dose of a Btk inhibitor; and administeringa second agent based on the determined expression profile.

In other embodiments, the present invention also provides methods fortreating a cancer comprising administering a Btk inhibitor sufficient toresult in an increase or appearance in the blood of a subpopulation oflymphocytes defined by immunophenotyping; and administering a secondagent once the increase or appearance in the blood of the subpopulationof lymphocytes is determined.

In some embodiments, the subject is a human.

In some embodiments, the Btk inhibitors are orally administered.

In any of the aforementioned aspects are further embodiments in whichadministration is enteral, parenteral, or both, and wherein (a) theeffective amount of the Btk inhibitor is systemically administered tothe mammal; (b) the effective amount of the Btk inhibitor isadministered orally to the mammal; (c) the effective amount of the Btkinhibitor is intravenously administered to the mammal; (d) the effectiveamount of the Btk inhibitor administered by inhalation; (e) theeffective amount of the Btk inhibitor is administered by nasaladministration; or (f) the effective amount of the Btk inhibitor isadministered by injection to the mammal; (g) the effective amount of theBtk inhibitor is administered topically (dermal) to the mammal; (h) theeffective amount of the Btk inhibitor is administered by ophthalmicadministration; or (i) the effective amount of the Btk inhibitor isadministered rectally to the mammal.

In any of the aforementioned aspects are further embodiments comprisingsingle administrations of the effective amount of the Btk inhibitor,including further embodiments in which (i) the Btk inhibitor isadministered once; (ii) the Btk inhibitor is administered to the mammalmultiple times over the span of one day; (iii) continually; or (iv)continuously.

In any of the aforementioned aspects are further embodiments comprisingmultiple administrations of the effective amount of the Btk inhibitor,including further embodiments in which (i) the Btk inhibitor isadministered in a single dose; (ii) the time between multipleadministrations is every 6 hours; (iii) the Btk inhibitor isadministered to the mammal every 8 hours. In further or alternativeembodiments, the method comprises a drug holiday, wherein theadministration of the Btk inhibitor is temporarily suspended or the doseof the Btk inhibitor being administered is temporarily reduced; at theend of the drug holiday, dosing of the Btk inhibitor is resumed. Thelength of the drug holiday can vary from 2 days to 1 year.

In any of the aforementioned aspects are further embodiments in whichadministration is enteral, parenteral, or both, and wherein (a) theeffective amount of the second agent is systemically administered to themammal; (b) the effective amount of the second agent is administeredorally to the mammal; (c) the effective amount of the second agent isintravenously administered to the mammal; (d) the effective amount ofthe second agent administered by inhalation; (e) the effective amount ofthe second agent is administered by nasal administration; or (f) theeffective amount of the second agent is administered by injection to themammal; (g) the effective amount of the second agent is administeredtopically (dermal) to the mammal; (h) the effective amount of the secondagent is administered by ophthalmic administration; or (i) the effectiveamount of the second agent is administered rectally to the mammal.

In any of the aforementioned aspects are further embodiments comprisingsingle administrations of the effective amount of second agent,including further embodiments in which (i) the second agent isadministered once; (ii) the second agent is administered to the mammalmultiple times over the span of one day; (iii) continually; or (iv)continuously.

In any of the aforementioned aspects are further embodiments comprisingmultiple administrations of the effective amount of the second agent,including further embodiments in which (i) the second agent isadministered in a single dose; (ii) the time between multipleadministrations is every 6 hours; (iii) the second agent is administeredto the mammal every 8 hours. In further or alternative embodiments, themethod comprises a drug holiday, wherein the administration of thesecond agent is temporarily suspended or the dose of the second agentbeing administered is temporarily reduced; at the end of the drugholiday, dosing of the second agent is resumed. The length of the drugholiday can vary from 2 days to 1 year.

\In any of the aforementioned aspects the second agent is selected fromthe group consisting of alemtuzumab, arsenic trioxide, asparaginase(pegylated or non-), bevacizumab, cetuximab, platinum-based compoundssuch as cisplatin, cladribine, daunorubicin/doxorubicin/idarubicin,irinotecan, fludarabine, 5-fluorouracil, gemtuzumab, methotrexate,Paclitaxel™, taxol, temozolomide, thioguanine, or classes of drugsincluding hormones (an antiestrogen, an antiandrogen, or gonadotropinreleasing hormone analogues, interferons such as alpha interferon,nitrogen mustards such as busulfan or melphalan or mechlorethamine,retinoids such as tretinoin, topoisomerase inhibitors such as irinotecanor topotecan, tyrosine kinase inhibitors such as gefinitinib orimatinib, or agents to treat signs or symptoms induced by such therapyincluding allopurinol, filgrastim, granisetron/ondansetron/palonosetron,dronabinol.

Preparation of Compounds

Compounds of Formula D may be synthesized using standard synthetictechniques known to those of skill in the art or using methods known inthe art in combination with methods described herein. In additions,solvents, temperatures and other reaction conditions presented hereinmay vary according to those of skill in the art. As a further guide thefollowing synthetic methods may also be utilized.

The reactions can be employed in a linear sequence to provide thecompounds described herein or they may be used to synthesize fragmentswhich are subsequently joined by the methods described herein and/orknown in the art.

Formation of Covalent Linkages by Reaction of an Electrophile with aNucleophile

The compounds described herein can be modified using variouselectrophiles or nucleophiles to form new functional groups orsubstituents. Table 1 entitled “Examples of Covalent Linkages andPrecursors Thereof” lists selected examples of covalent linkages andprecursor functional groups which yield and can be used as guidancetoward the variety of electrophiles and nucleophiles combinationsavailable. Precursor functional groups are shown as electrophilic groupsand nucleophilic groups.

TABLE 1 Examples of Covalent Linkages and Precursors Thereof CovalentLinkage Product Electrophile Nucleophile Carboxamides Activated estersamines/anilines Carboxamides acyl azides amines/anilines Carboxamidesacyl halides amines/anilines Esters acyl halides alcohols/phenols Estersacyl nitriles alcohols/phenols Carboxamides acyl nitrilesamines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes orketones Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkylamines alkyl halides amines/anilines Esters alkyl halides carboxylicacids Thioethers alkyl halides Thiols Ethers alkyl halidesalcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkylsulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenolsEsters Anhydrides alcohols/phenols Carboxamides Anhydridesamines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halidesAmines Thioethers Azindines Thiols Boronate esters Boronates GlycolsCarboxamides carboxylic acids amines/anilines Esters carboxylic acidsAlcohols hydrazines Hydrazides carboxylic acids N-acylureas orAnhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylicacids Thioethers Epoxides Thiols Thioethers haloacetamides ThiolsAmmotriazines halotriazines amines/anilines Triazinyl ethershalotriazines alcohols/phenols Amidines imido esters amines/anilinesUreas Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenolsThioureas isothiocyanates amines/anilines Thioethers Maleimides ThiolsPhosphite esters phosphoramidites Alcohols Silyl ethers silyl halidesAlcohols Alkyl amines sulfonate esters amines/anilines Thioetherssulfonate esters Thiols Esters sulfonate esters carboxylic acids Etherssulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilinesSulfonate esters sulfonyl halides phenols/alcohols Alkyl thiolα,β-unsaturated ester thiols Alkyl ethers α,β-unsaturated ester alcoholsAlkyl amines α,β-unsaturated ester amines Alkyl thiol Vinyl sulfonethiols Alkyl ethers Vinyl sulfone alcohols Alkyl amines Vinyl sulfoneamines Vinyl sulfide Propargyl amide thiolUse of Protecting Groups

In the reactions described, it may be necessary to protect reactivefunctional groups, for example hydroxy, amino, imino, thio or carboxygroups, where these are desired in the final product, to avoid theirunwanted participation in the reactions. Protecting groups are used toblock some or all reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In one embodiment, each protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval. Protective groups can be removed by acid, base, andhydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and may be used to protect carboxyand hydroxy reactive moieties in the presence of amino groups protectedwith Cbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties maybe blocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl in the presence of amines blocked with acid labilegroups such as t-butyl carbamate or with carbamates that are both acidand base stable but hydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be protected by conversion to simple ester compounds as exemplifiedherein, or they may be blocked with oxidatively-removable protectivegroups such as 2,4-dimethoxybenzyl, while co-existing amino groups maybe blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in then presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with a Pd-catalyzedreaction in the presence of acid labile t-butyl carbamate or base-labileacetate amine protecting groups. Yet another form of protecting group isa resin to which a compound or intermediate may be attached. As long asthe residue is attached to the resin, that functional group is blockedand cannot react. Once released from the resin, the functional group isavailable to react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, and Kocienski,Protective Groups, Thieme Verlag, New York, N.Y., 1994, which areincorporated herein by reference in their entirety.

Further Forms of Compounds

The compounds described herein may possess one or more stereocenters andeach center may exist in the R or S configuration. The compoundspresented herein include all diastereomeric, enantiomeric, and epimericforms as well as the appropriate mixtures thereof. Stereoisomers may beobtained, if desired, by methods known in the art as, for example, theseparation of stereoisomers by chiral chromatographic columns.

Diasteromeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods known, for example, by chromatography and/or fractionalcrystallization. In one embodiment, enantiomers can be separated bychiral chromatographic columns. In other embodiments, enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. All such isomers, including diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein.

The methods and formulations described herein include the use ofN-oxides, crystalline forms (also known as polymorphs), orpharmaceutically acceptable salts of compounds described herein, as wellas active metabolites of these compounds having the same type ofactivity. In some situations, compounds may exist as tautomers. Alltautomers are included within the scope of the compounds presentedherein. In addition, the compounds described herein can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

Compounds of Formula D in unoxidized form can be prepared from N-oxidesof compounds of Formula D by treating with a reducing agent, such as,but not limited to, sulfur, sulfur dioxide, triphenyl phosphine, lithiumborohydride, sodium borohydride, phosphorus trichloride, tribromide, orthe like in a suitable inert organic solvent, such as, but not limitedto, acetonitrile, ethanol, aqueous dioxane, or the like at 0 to 80° C.

In some embodiments, compounds described herein are prepared asprodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a compound described herein, which isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is detrimental to mobilitybut which then is metabolically hydrolyzed to the carboxylic acid, theactive entity, once inside the cell where water-solubility isbeneficial. A further example of a prodrug might be a short peptide(polyaminoacid) bonded to an acid group where the peptide is metabolizedto reveal the active moiety. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Incertain embodiments, a prodrug is enzymatically metabolized by one ormore steps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, apharmaceutically active compound is modified such that the activecompound will be regenerated upon in vivo administration. The prodrugcan be designed to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound. (see, forexample, Nogrady (1985) Medicinal Chemistry A Biochemical Approach,Oxford University Press, New York, pages 388-392; Silverman (1992), TheOrganic Chemistry of Drug Design and Drug Action, Academic Press, Inc.,San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic andMedicinal Chemistry Letters, Vol. 4, p. 1985).

Prodrug forms of the herein described compounds, wherein the prodrug ismetabolized in vivo to produce a derivative as set forth herein areincluded within the scope of the claims. In some cases, some of theherein-described compounds may be a prodrug for another derivative oractive compound.

Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. Prodrugs may be designed as reversible drugderivatives, for use as modifiers to enhance drug transport tosite-specific tissues. In some embodiments, the design of a prodrugincreases the effective water solubility. See, e.g., Fedorak et al., Am.J. Physiol., 269:G210-218 (1995); McLoed et al., Gastroenterol,106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992);J. Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987); J.Larsen et al., Int. J. Pharmaceutics, 47, 103 (1988); Sinkula et al., J.Pharm. Sci., 64:181-210 (1975); T. Higuchi and V. Stella, Pro-drugs asNovel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; andEdward B. Roche, Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, all incorporatedherein in their entirety.

Sites on the aromatic ring portion of compounds of Formula D can besusceptible to various metabolic reactions, therefore incorporation ofappropriate substituents on the aromatic ring structures, such as, byway of example only, halogens can reduce, minimize or eliminate thismetabolic pathway.

Compounds described herein include isotopically-labeled compounds, whichare identical to those recited in the various formulas and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labeled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

In additional or further embodiments, the compounds described herein aremetabolized upon administration to an organism in need to produce ametabolite that is then used to produce a desired effect, including adesired therapeutic effect.

Compounds described herein may be formed as, and/or used as,pharmaceutically acceptable salts. The type of pharmaceutical acceptablesalts, include, but are not limited to: (1) acid addition salts, formed)by reacting the free base form of the compound with a pharmaceuticallyacceptable: inorganic acid such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, andthe like; or with an organic acid such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoicacid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonicacid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; (2) salts formed when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium),an alkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion;or coordinates with an organic base. Acceptable organic bases includeethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases includealuminum hydroxide, calcium hydroxide, potassium hydroxide, sodiumcarbonate, sodium hydroxide, and the like.

The corresponding counterions of the pharmaceutically acceptable saltsmay be analyzed and identified using various methods including, but notlimited to, ion exchange chromatography, ion chromatography, capillaryelectrophoresis, inductively coupled plasma, atomic absorptionspectroscopy, mass spectrometry, or any combination thereof.

The salts are recovered by using at least one of the followingtechniques: filtration, precipitation with a non-solvent followed byfiltration, evaporation of the solvent, or, in the case of aqueoussolutions, lyophilization.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms or crystal formsthereof, particularly solvates or polymorphs. Solvates contain eitherstoichiometric or non-stoichiometric amounts of a solvent, and may beformed during the process of crystallization with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. Hydrates areformed when the solvent is water, or alcoholates are formed when thesolvent is alcohol. Solvates of compounds described herein can beconveniently prepared or formed during the processes described herein.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol, and the like. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate.

Compounds described herein may be in various forms, including but notlimited to, amorphous forms, milled forms and nano-particulate forms. Inaddition, compounds described herein include crystalline forms, alsoknown as polymorphs. Polymorphs include the different crystal packingarrangements of the same elemental composition of a compound. Polymorphsusually have different X-ray diffraction patterns, infrared spectra,melting points, density, hardness, crystal shape, optical and electricalproperties, stability, and solubility. Various factors such as therecrystallization solvent, rate of crystallization, and storagetemperature may cause a single crystal form to dominate.

The screening and characterization of the pharmaceutically acceptablesalts, polymorphs and/or solvates may be accomplished using a variety oftechniques including, but not limited to, thermal analysis, x-raydiffraction, spectroscopy, vapor sorption, and microscopy. Thermalanalysis methods address thermo chemical degradation or thermo physicalprocesses including, but not limited to, polymorphic transitions, andsuch methods are used to analyze the relationships between polymorphicforms, determine weight loss, to find the glass transition temperature,or for excipient compatibility studies. Such methods include, but arenot limited to, Differential scanning calorimetry (DSC), ModulatedDifferential Scanning calorimetry (MDCS), Thermogravimetric analysis(TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-raydiffraction methods include, but are not limited to, single crystal andpowder diffractometers and synchrotron sources. The variousspectroscopic techniques used include, but are not limited to, Raman,FTIR, UVIS, and NMR (liquid and solid state). The various microscopytechniques include, but are not limited to, polarized light microscopy,Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis(EDX), Environmental Scanning Electron Microscopy with EDX (in gas orwater vapor atmosphere), IR microscopy, and Raman microscopy.

Throughout the specification, groups and substituents thereof can bechosen by one skilled in the field to provide stable moieties andcompounds.

Cancer Treatment Regimens

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisesmeasuring the peripheral blood concentration of the mobilized pluralityof cells. In some embodiments, the method further comprisesadministering a second cancer treatment regimen after the peripheralblood concentration of the mobilized plurality of cells increases ascompared to the concentration before administration of the Btkinhibitor. In some embodiments, administering the second cancertreatment regimen occurs after a subsequent decrease in peripheral bloodconcentration of the mobilized plurality of cells. In some embodiments,analyzing the mobilized plurality of cells comprises measuring theduration of an increase in the peripheral blood concentration of themobilized plurality of cells as compared to the concentration beforeadministration of the Btk inhibitor. In some embodiments, the methodfurther comprises administering a second cancer treatment regimen afterthe peripheral blood concentration of the mobilized plurality of cellshas increased for a predetermined length of time. In some embodiments,analyzing the mobilized plurality of cells comprises counting the numberof mobilized plurality of cells in the peripheral blood. In someembodiments, the method further comprises administering a second cancertreatment regimen after the number of mobilized plurality of cells inthe peripheral blood increases as compared to the number beforeadministration of the Btk inhibitor. In some embodiments, administeringthe second cancer treatment regimen occurs after a subsequent decreasein the number of mobilized plurality of cells in the peripheral blood.In some embodiments, analyzing the mobilized plurality of cellscomprises measuring the duration of an increase in the number ofmobilized plurality of cells in the peripheral blood as compared to thenumber before administration of the Btk inhibitor. In some embodiments,the method further comprises administering a second cancer treatmentregimen after the number of mobilized plurality of cells in theperipheral blood has increased for a predetermined length of time.

In some embodiments, administering a Btk inhibitor before a secondcancer treatment regimen reduces immune-mediated reactions to the secondcancer treatment regimen. In some embodiments, administering a Btkinhibitor before ofatumumab reduces immune-mediated reactions toofatumumab.

In some embodiments, the second cancer treatment regimen comprises achemotherapeutic agent, a steroid, an immunotherapeutic agent, atargeted therapy, or a combination thereof. In some embodiments, thesecond cancer treatment regimen comprises a B cell receptor pathwayinhibitor. In some embodiments, the B cell receptor pathway inhibitor isa CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor,a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, aPKCβ inhibitor, or a combination thereof. In some embodiments, thesecond cancer treatment regimen comprises an antibody, B cell receptorsignaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTORinhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosomeinhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor,a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, aJak1/2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARPinhibitor, or a combination thereof.

In some embodiments, the second cancer treatment regimen compriseschlorambucil, ifosphamide, doxorubicin, mesalazine, thalidomide,lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib,paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone,CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin,or a combination thereof.

In some embodiments, the second cancer treatment regimen comprisescyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, andoptionally, rituximab.

In some embodiments, the second cancer treatment regimen comprisesbendamustine, and rituximab.

In some embodiments, the second cancer treatment regimen comprisesfludarabine, cyclophosphamide, and rituximab.

In some embodiments, the second cancer treatment regimen comprisescyclophosphamide, vincristine, and prednisone, and optionally,rituximab.

In some embodiments, the second cancer treatment regimen comprisesetoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, andoptionally, rituximab.

In some embodiments, the second cancer treatment regimen comprisesdexamethasone and lenalidomide.

Additional cancer treatment regimens include Nitrogen Mustards such asfor example, bendamustine, chlorambucil, chlormethine, cyclophosphamide,ifosfamide, melphalan, prednimustine, trofosfamide; Alkyl Sulfonateslike busulfan, mannosulfan, treosulfan; Ethylene Imines like carboquone,thiotepa, triaziquone; Nitrosoureas like carmustine, fotemustine,lomustine, nimustine, ranimustine, semustine, streptozocin; Epoxidessuch as for example, etoglucid; Other Alkylating Agents such as forexample dacarbazine, mitobronitol, pipobroman, temozolomide; Folic AcidAnalogues such as for example methotrexate, permetrexed, pralatrexate,raltitrexed; Purine Analogs such as for example cladribine, clofarabine,fludarabine, mercaptopurine, nelarabine, tioguanine; Pyrimidine Analogssuch as for example azacitidine, capecitabine, carmofur, cytarabine,decitabine, fluorouracil, gemcitabine, tegafur; Vinca Alkaloids such asfor example vinblastine, vincristine, vindesine, vinflunine,vinorelbine; Podophyllotoxin Derivatives such as for example etoposide,teniposide; Colchicine derivatives such as for example demecolcine;Taxanes such as for example docetaxel, paclitaxel, paclitaxelpoliglumex; Other Plant Alkaloids and Natural Products such as forexample trabectedin; Actinomycines such as for example dactinomycin;Antracyclines such as for example aclarubicin, daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin,valrubicin, zorubincin; Other Cytotoxic Antibiotics such as for examplebleomycin, ixabepilone, mitomycin, plicamycin; Platinum Compounds suchas for example carboplatin, cisplatin, oxaliplatin, satraplatin;Methylhydrazines such as for example procarbazine; Sensitizers such asfor example aminolevulinic acid, efaproxiral, methyl aminolevulinate,porfimer sodium, temoporfin; Protein Kinase Inhibitors such as forexample dasatinib, erlotinib, everolimus, gefitinib, imatinib,lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus;Other Antineoplastic Agents such as for example alitretinoin,altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase,bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine,hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein,mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin,romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin,vorinostat; Estrogens such as for example diethylstilbenol,ethinylestradiol, fosfestrol, polyestradiol phosphate; Progestogens suchas for example gestonorone, medroxyprogesterone, megestrol; GonadotropinReleasing Hormone Analogs such as for example buserelin, goserelin,leuprorelin, triptorelin; Anti-Estrogens such as for examplefulvestrant, tamoxifen, toremifene; Anti-Androgens such as for examplebicalutamide, flutamide, nilutamide, Enzyme Inhibitors,aminoglutethimide, anastrozole, exemestane, formestane, letrozole,vorozole; Other Hormone Antagonists such as for example abarelix,degarelix; Immunostimulants such as for example histaminedihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex,thymopentin; Immunosuppressants such as for example everolimus,gusperimus, leflunomide, mycophenolic acid, sirolimus; CalcineurinInhibitors such as for example ciclosporin, tacrolimus; OtherImmunosuppressants such as for example azathioprine, lenalidomide,methotrexate, thalidomide; and Radiopharmaceuticals such as for example,iobenguane.

Additional cancer treatment regimens include interferons, interleukins,Tumor Necrosis Factors, Growth Factors, or the like.

Additional cancer treatment regimens include Immuno stimulants such asfor example ancestim, filgrastim, lenograstim, molgramostim,pegfilgrastim, sargramostim; Interferons such as for example interferonalfa natural, interferon alfa-2a, interferon alfa-2b, interferonalfacon-1, interferon alfa-n1, interferon beta natural, interferonbeta-1a, interferon beta-1b, interferon gamma, peginterferon alfa-2a,peginterferon alfa-2b; Interleukins such as for example aldesleukin,oprelvekin; Other Immunostimulants such as for example BCG vaccine,glatiramer acetate, histamine dihydrochloride, immunocyanin, lentinan,melanoma vaccine, mifamurtide, pegademase, pidotimod, plerixafor, polyI:C, poly ICLC, roquinimex, tasonermin, thymopentin; Immunosuppressantssuch as for example abatacept, abetimus, alefacept, antilymphocyteimmunoglobulin (horse), antithymocyte immunoglobulin (rabbit),eculizumab, efalizumab, everolimus, gusperimus, leflunomide,muromab-CD3, mycophenolic acid, natalizumab, sirolimus; TNF alphaInhibitors such as for example adalimumab, afelimomab, certolizumabpegol, etanercept, golimumab, infliximab; Interleukin Inhibitors such asfor example anakinra, basiliximab, canakinumab, daclizumab, mepolizumab,rilonacept, tocilizumab, ustekinumab; Calcineurin Inhibitors such as forexample ciclosporin, tacrolimus; Other Immunosuppressants such as forexample azathioprine, lenalidomide, methotrexate, thalidomide.

Additional cancer treatment regimens include Adalimumab, Alemtuzumab,Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol, Daclizumab,Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab,Muromonab-CD3, Natalizumab, Panitumumab, Ranibizumab, Rituximab,Tositumomab, Trastuzumab, or the like, or a combination thereof.

Additional cancer treatment regimens include Monoclonal Antibodies suchas for example alemtuzumab, bevacizumab, catumaxomab, cetuximab,edrecolomab, gemtuzumab, ofatumumab, panitumumab, rituximab,trastuzumab, Immunosuppressants, eculizumab, efalizumab, muromab-CD3,natalizumab; TNF alpha Inhibitors such as for example adalimumab,afelimomab, certolizumab pegol, golimumab, infliximab, InterleukinInhibitors, basiliximab, canakinumab, daclizumab, mepolizumab,tocilizumab, ustekinumab, Radiopharmaceuticals, ibritumomab tiuxetan,tositumomab; Others Monoclonal Antibodies such as for exampleabagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonal antibodyXmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab,arcitumomab, basiliximab, bispecific antibody 2B1, blinatumomab,brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab,conatumumab, dacetuzumab, denosumab, eculizumab, epratuzumab,epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab,galiximab, ganitumab, gemtuzumab ozogamicin, glembatumumab, ibritumomab,inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab,lucatumumab, mapatumumab, matuzumab, milatuzumab, monoclonal antibodyCC49, necitumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab,ramacurimab, ranibizumab, siplizumab, sonepcizumab, tanezumab,tositumomab, trastuzumab, tremelimumab, tucotuzumab celmoleukin,veltuzumab, visilizumab, volociximab, zalutumumab.

Additional cancer treatment regimens include agents that affect thetumor micro-environment such as cellular signaling network (e.g.phosphatidylinositol 3-kinase (PI3K) signaling pathway, signaling fromthe B-cell receptor and the IgE receptor). In some embodiments, thesecond agent is a PI3K signaling inhibitor or a syc kinase inhibitor. Inone embodiment, the syk inhibitor is R788. In another embodiment is aPKCγ inhibitor such as by way of example only, enzastaurin.

Examples of agents that affect the tumor micro-environment include PI3Ksignaling inhibitor, syc kinase inhibitor, Protein Kinase Inhibitorssuch as for example dasatinib, erlotinib, everolimus, gefitinib,imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib,temsirolimus; Other Angiogenesis Inhibitors such as for example GT-111,JI-101, R1530; Other Kinase Inhibitors such as for example AC220, AC480,ACE-041, AMG 900, AP24534, Arry-614, AT7519, AT9283, AV-951, axitinib,AZD1152, AZD7762, AZD8055, AZD8931, bafetinib, BAY 73-4506, BGJ398,BGT226, BI 811283, BI6727, BIBF 1120, BIBW 2992, BMS-690154, BMS-777607,BMS-863233, BSK-461364, CAL-101, CEP-11981, CYC116, DCC-2036,dinaciclib, dovitinib lactate, E7050, EMD 1214063, ENMD-2076,fostamatinib disodium, GSK2256098, GSK690693, INCB18424, INNO-406,JNJ-26483327, JX-594, KX2-391, linifanib, LY2603618, MGCD265, MK-0457,MK1496, MLN8054, MLN8237, MP470, NMS-1116354, NMS-1286937, ON 01919.Na,OSI-027, OSI-930, Btk inhibitor, PF-00562271, PF-02341066, PF-03814735,PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397,progenipoietin, R547, R763, ramucirumab, regorafenib, RO5185426,SAR103168, SCH 727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901,TKI258, TLN-232, TTP607, XL147, XL228, XL281RO5126766, XL418, XL765.

Further examples of anti-cancer agents for use in combination with a Btkinhibitor compound include inhibitors of mitogen-activated proteinkinase signaling, e.g., U0126, PD98059, PD184352, PD0325901,ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002;Syk inhibitors; mTOR inhibitors; and antibodies (e.g., rituxan).

Other anti-cancer agents that can be employed in combination with a Btkinhibitor compound include Adriamycin, Dactinomycin, Bleomycin,Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin Il (includingrecombinant interleukin II, or rlL2), interferon alfa-2a; interferonalfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-1 a;interferon gamma-1 b; iproplatin; irinotecan hydrochloride; lanreotideacetate; letrozole; leuprolide acetate; liarozole hydrochloride;lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;maytansine; mechlorethamine hydrochloride; megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other anti-cancer agents that can be employed in combination with a Btkinhibitor compound include: 20-epi-1, 25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-such asfor example growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

Yet other anticancer agents that can be employed in combination with aBtk inhibitor compound include alkylating agents, antimetabolites,natural products, or hormones, e.g., nitrogen mustards (e.g.,mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne,ete.), or triazenes (decarbazine, etc.). Examples of antimetabolitesinclude but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin).

Examples of alkylating agents that can be employed in combination a Btkinhibitor compound include, but are not limited to, nitrogen mustards(e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan,etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes(decarbazine, ete.). Examples of antimetabolites include, but are notlimited to folic acid analog (e.g., methotrexate), or pyrimidine analogs(e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin.

Examples of anti-cancer agents which act by arresting cells in the G2-Mphases due to stabilized microtubules and which can be used incombination with a Btk inhibitor compound include without limitation thefollowing marketed drugs and drugs in development: Erbulozole (alsoknown as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128),Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829,Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also knownas E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C),Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3,Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7,Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also knownas LU-103793 and NSC-D-669356), Epothilones (such as Epothilone A,Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA),Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothiloneB), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone AN-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known asBMS-310705), 21-hydroxyepothilone D (also known as Desoxyepothilone Fand dEpoF), 26-fluoroepothilone), Auristatin PE (also known asNSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia,also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P),LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis),Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also knownas WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academyof Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651),SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97(Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739(Ajinomoto, also known as AVE-8063A and CS-39.HCI), AC-7700 (Ajinomoto,also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCI, and RPR-258062A),Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known asNSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 andTI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 andWHI-261), H10 (Kansas State University), H16 (Kansas State University),Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker HughesInstitute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute),SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569),Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica),A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai Schoolof Medicine, also known as MF-191), TMPN (Arizona State University),Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine(also known as NSC-698666), 3-1AABE (Cytoskeleton/Mt. Sinai School ofMedicine), A-204197 (Abbott), T-607 (Tuiarik, also known as T-900607),RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin,Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin),Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica),D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350(Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott),Diozostatin, (−)-Phenylahistin (also known as NSCL-96F037), D-68838(Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris,also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286(also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317(Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphatesodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411(Sanofi).

Biomarkers

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, analyzing the mobilized plurality of cells comprisespreparing a biomarker profile for a population of cells isolated fromthe plurality of cells. In some embodiments, the biomarker expressionprofile is used to diagnose, determine a prognosis, or create apredictive profile of a hematological malignancy. In some embodiments,the biomarker profile indicates the expression of a biomarker, theexpression level of a biomarker, mutations in a biomarker, or thepresence of a biomarker. In some embodiments, the biomarker is anycytogenetic, cell surface molecular or protein or RNA expression marker.In some embodiments, the biomarker is: ZAP70; t(14,18); β-2microglobulin; p53 mutational status; ATM mutational status; del(17)p;del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25; CD38; CD103;CD138; secreted, surface or cytoplasmic immunoglobulin expression; V_(H)mutational status; or a combination thereof. In some embodiments, themethod further comprises providing a second cancer treatment regimenbased on the biomarker profile. In some embodiments, the method furthercomprises not administering based on the biomarker profile. In someembodiments, the method further comprises predicting the efficacy of atreatment regimen based on the biomarker profile.

In certain embodiments, the methods comprise diagnosing, determining aprognosis, or creating a predictive profile of a hematologicalmalignancy based upon the expression or presence of certain biomarkers.In other embodiments, the methods further comprise stratifying patientpopulations based upon the expression or presence of certain biomarkersin the affected lymphocytes. In still other embodiments, the methodsfurther comprise determining a therapeutic regimen for the subject basedupon the expression or presence of certain biomarkers in the affectedlymphocytes. In yet other embodiments, the methods further comprisepredicting a response to therapy in a subject based upon the expressionor presence of certain biomarkers in the affected lymphocytes.

In certain aspects, provided herein are methods of diagnosing,determining a prognosis, or creating a predictive profile of ahematological malignancy in a subject comprising: (a) administering aBtk inhibitor to the subject sufficient to result in an increase orappearance in the blood of a subpopulation of lymphocytes; and (b)determining the expression or presence of one or more biomarkers fromone or more subpopulation of lymphocytes; wherein the expression orpresence of one or more biomarkers is used to diagnose the hematologicalmalignancy, determine the prognosis of the hematological malignancy, orcreate a predictive profile of the hematological malignancy. In oneembodiment, the increase or appearance in the blood of a subpopulationof lymphocytes is determined by immunophenotyping. In anotherembodiment, the increase or appearance in the blood of a subpopulationof lymphocytes is determined by fluorescent activated cell sorting(FACS).

In other aspects, provided herein are methods of stratifying a patientpopulation having a hematological malignancy comprising: (a)administering a Btk inhibitor to the subject sufficient to result in anincrease or appearance in the blood of a subpopulation of lymphocytes;and (b) determining the expression or presence of one or more biomarkersfrom one or more subpopulation of lymphocytes; wherein the expression orpresence of one or more biomarkers is used to stratify patients fortreatment of the hematological malignancy. In one embodiment, theincrease or appearance in the blood of a subpopulation of lymphocytes isdetermined by immunophenotyping. In another embodiment, the increase orappearance in the blood of a subpopulation of lymphocytes is determinedby fluorescent activated cell sorting (FACS).

In still other aspects, provided herein are methods of determining atherapeutic regimen in a subject having a hematological malignancycomprising: (a) administering a Btk inhibitor to the subject sufficientto result in an increase or appearance in the blood of a subpopulationof lymphocytes; and (b) determining the expression or presence of one ormore biomarkers from one or more subpopulation of lymphocytes; whereinthe expression or presence of one or more biomarkers is used todetermine the therapeutic regimen for the treatment of the hematologicalmalignancy. In one embodiment, the increase or appearance in the bloodof a subpopulation of lymphocytes is determined by immunophenotyping. Inanother embodiment, the increase or appearance in the blood of asubpopulation of lymphocytes is determined by fluorescent activated cellsorting (FACS).

In yet other aspects, provided herein are methods of predicting aresponse to therapy in a subject having a hematological malignancycomprising: (a) administering a Btk inhibitor to the subject sufficientto result in an increase or appearance in the blood of a subpopulationof lymphocytes; and (b) determining the expression or presence of one ormore biomarkers from one or more subpopulation of lymphocytes; whereinthe expression or presence of one or more biomarkers is used to predictthe subject's response to therapy for the hematological malignancy. Inone embodiment, the increase or appearance in the blood of asubpopulation of lymphocytes is determined by immunophenotyping. Inanother embodiment, the increase or appearance in the blood of asubpopulation of lymphocytes is determined by fluorescent activated cellsorting (FACS).

In certain aspects, provided herein are methods of diagnosing,determining a prognosis, or creating a predictive profile of ahematological malignancy in a subject comprising determining theexpression or presence of one or more biomarkers from one or moresubpopulation of lymphocytes in a subject that has received a dose of aBtk inhibitor wherein the expression or presence of one or morebiomarkers is used to diagnose the hematological malignancy, determinethe prognosis of the hematological malignancy, or create a predictiveprofile of the hematological malignancy. In one embodiment, the dose ofBtk inhibitor is sufficient to result in an increase or appearance inthe blood of a subpopulation of lymphocytes defined byimmunophenotyping. In another embodiment, the determining the expressionor presence of one or more biomarkers from one or more subpopulation oflymphocytes further comprises isolating, detecting or measuring one ormore type of lymphocyte. In still another embodiment, the Btk inhibitoris a reversible or irreversible inhibitor.

In other aspects, provided herein are methods of stratifying a patientpopulation having a hematological malignancy comprising determining theexpression or presence of one or more biomarkers from one or moresubpopulation of lymphocytes in a subject that has received a dose of aBtk inhibitor wherein the expression or presence of one or morebiomarkers is used to stratify patients for treatment of thehematological malignancy. In one embodiment, the dose of Btk inhibitoris sufficient to result in an increase or appearance in the blood of asubpopulation of lymphocytes defined by immunophenotyping. In anotherembodiment, the determining the expression or presence of one or morebiomarkers from one or more subpopulation of lymphocytes furthercomprises isolating, detecting or measuring one or more type oflymphocyte. In still another embodiment, the Btk inhibitor is areversible or irreversible inhibitor.

In still other aspects, provided herein are methods of determining thetherapeutic regimen in a subject having a hematological malignancycomprising determining the expression or presence of one or morebiomarkers from one or more subpopulation of lymphocytes in a subjectthat has received a dose of a Btk inhibitor wherein the expression orpresence of one or more biomarkers is used to determine the therapeuticregimen for the treatment of the hematological malignancy. In oneembodiment, the dose of Btk inhibitor is sufficient to result in anincrease or appearance in the blood of a subpopulation of lymphocytesdefined by immunophenotyping. In another embodiment, the determining theexpression or presence of one or more biomarkers from one or moresubpopulation of lymphocytes further comprises isolating, detecting ormeasuring one or more type of lymphocyte. In still another embodiment,the Btk inhibitor is a reversible or irreversible inhibitor.

In yet other aspects, provided herein are methods of predicting aresponse to therapy in a subject having a hematological malignancycomprising determining the expression or presence of one or morebiomarkers from one or more circulating lymphocytes in a subject thathas received a dose of a Btk inhibitor wherein the expression orpresence of one or more biomarkers is used to predict the subject'sresponse to therapy for the hematological malignancy. In one embodiment,the dose of Btk inhibitor is sufficient to result in an increase orappearance in the blood of a subpopulation of lymphocytes defined byimmunophenotyping. In another embodiment, the determining the expressionor presence of one or more biomarkers from one or more subpopulation oflymphocytes further comprises isolating, detecting or measuring one ormore type of lymphocyte. In still another embodiment, the Btk inhibitoris a reversible or irreversible inhibitor.

As contemplated herein, any biomarker related to hematologicalmalignancies are in some embodiments utilized in the present methods.These biomarkers include any biological molecule (found either in blood,other body fluids, or tissues) or any chromosomal abnormality that is asign of a hematological malignancy. In certain embodiments, thebiomarkers include, but are not limited to, TdT, CD5, CD11c, CD19, CD20,CD22, CD79a, CD15, CD30, CD38, CD138, CD103, CD25, ZAP-70, p53mutational status, ATM mutational status, mutational status of IgV_(H),chromosome 17 deletions (del 17p), chromosome 6 deletions (del 6q),chromosome 7 deletions (del 7q), chromosome 11 deletions (del 11q),trisomy 12, chromosome 13 deletions (del 13 q), t(11:14) chromosomaltranslocation, t(14:18) chromosomal translocation, CD10, CD23, beta-2microglobulin, bcl-2 expression, CD9, presence of Helicobacter pylori,CD154/CD40, Akt, NF-κB, WNT, Mtor, ERK, MAPK, and Src tyrosine kinaseexpression. In certain embodiments, the biomarkers include ZAP-70, CD5,t(14;18), CD38, β-2 microglobulin, p53 mutational status, ATM mutationalstatus, chromosome 17p deletion, chromosome 11q deletion, surface orcytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22,CD11c, CD 103, chromosome 7q deletion, V_(H) mutational status, or acombination thereof.

In certain embodiments, subpopulations of patients having ahematological malignancy cancer or pre-that would benefit from a knowntreatment regimen are identified by screening candidate subjects for oneor more clinically useful biomarkers known in the art. Any clinicallyuseful prognostic marker known to those of skill in the art can be used.In some embodiments, the subpopulation includes patients having chroniclymphocytic leukemia (CLL), and the clinically useful prognostic markersof particular interest include, but are not limited to, ZAP-70, CD38,.beta.2 microglobulin, and cytogenetic markers, for example, p53mutational status, ATM mutational status, chromosome deletions, such asthe chromosome 17p deletion and the chromosome 11q deletion, all ofwhich are clinically useful prognostic markers for this disease.

ZAP-70 is a tyrosine kinase that associates with the zeta subunit of theT cell antigen receptor (TCR) and plays a pivotal role in T cellactivation and development (Chan et al. (1992) Cell 71:649-662). ZAP-70undergoes tyrosine phosphorylation and is essential in mediating signaltransduction following TCR stimulation. Overexpression or constitutiveactivation of tyrosine kinases has been demonstrated to be involved in anumber of malignancies including leukemias and several types of solidtumors. For example, increased ZAP-70 RNA expression levels are aprognostic marker of chronic lymphocytic leukemia (CLL) (Rosenwald etal. (2001) J. Exp. Med. 194:1639-1647). ZAP-70 is expressed in T-cellsand natural killer cells, but is not known to be expressed in normalB-cells. However, ZAP-70 is expressed at high levels in the B-cells ofchronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL)patients, and more particularly in the subset of CLL patients who tendto have the more aggressive clinical course that is found in CLL/SLLpatients with unmutated Ig genes (Wiestner et al. (2003) Blood 101:4944-4951; U.S. Patent Application Publication No. 20030203416). Becauseof the correlation between ZAP-70 expression levels and Ig gene mutationstatus, ZAP-70 can be used as a prognostic indicator to identify thosepatients likely to have severe disease (high ZAP-70, unmutated Iggenes), and who are therefore candidates for aggressive therapy.

CD38 is a signal transduction molecule as well as an ectoenzymecatalyzing the synthesis and degradation of cyclic ADP ribose (cADPR).CD38 expression is present at high levels in bone marrow precursor Bcells, is down-regulated in resting normal B cells, and then isre-expressed in terminally differentiated plasma cells (Campana et al.(2000) Chem. Immunol. 75:169-188). CD38 is a reliable prognosticindicator in B-CLL, with the expression of CD38 generally indicating aless favorable outcome (D'Arena et al. (2001) Leuk. Lymphoma 42:109; DelPoeta et al. (2001) Blood 98:2633; Durig et al. (2002) Leukemia 16:30;Ibrahim et al. (2001) Blood 98:181; Deaglio et al. (2003) Blood102:2146-2155). The unfavorable clinical indications that CD38expression has been associated with include an advanced stage ofdisease, poor responsiveness to chemotherapy, a shorter time beforeinitial treatment is required, and a shorter survival time (Deaglio etal. (2003) Blood 102:2146-2155). Initially, a strong correlation betweenCD38 expression and IgV gene mutation was observed, with patients havingunmutated V genes displaying higher percentages of CD38.sup.+B-CLL cellsthan those with mutated V genes (Damle et al. (1999) Blood94:1840-1847). However, subsequent studies have indicated that CD38expression does not always correlate with the rearrangement of the IgVgenes (Hamblin et al. (2002) Blood 99:1023; Thunberg et al. (2001) Blood97:1892).

p53 is a nuclear phosphoprotein that acts as a tumor suppressor.Wild-type p53 is involved in regulating cell growth and division. p53binds to DNA, stimulating the production of a protein (p21) thatinteracts with a cell division-stimulating protein (cdk2). When p21 isbound to cdk2, the cell is blocked from entering the next stage of celldivision. Mutant p53 is incapable of binding DNA effectively, thuspreventing p21 from acting as the stop signal for cell division,resulting in uncontrolled cell division, and tumor formation. p53 alsoregulates the induction of programmed cell death (apoptosis) in responseto DNA damage, cell stress or the aberrant expression of some oncogenes.Expression of wild type p53 in some cancer cell lines has been shown torestore growth suppression control (Casey et al. (1991) Oncogene6:1791-1797; Takahashi et al. (1992) Cancer Res. 52:734-736). Mutationsin p53 are found in most tumor types, including tumors of the colon,breast, lung, ovary, bladder, and many other organs. p53 mutations havebeen found to be associated with Burkitt's lymphoma, L3-type B-cellacute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia(Gaidano et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5413-5417). p53abnormalities have also been found associated with B-cell prolymphocyticleukemia (Lens et al. (1997) Blood 89:2015-2023). The gene for p53 islocated on the short arm of chromosome 17 at 17p13.105-p12.

B-2-microglobulin is an extracellular protein that is noncovalentlyassociated with the .alpha. chain of the class I majorhistocompatibility complex (MHC). It is detectable in the serum, and isan adverse prognostic indicator in CLL (Keating et al. (1998) Blood86:606a) and Hodgkin's lymphoma (Chronowski et al. (2002) Cancer95:2534-2538). It is clinically used for lymphoproliferative diseasesincluding leukemia, lymphoma, and multiple myeloma, where serumβ-2-microglobulin levels are related to tumor cell load, prognosis, anddisease activity (Bataille et al. (1983) Br. J. Haematol. 55:439-447;Aviles et al. (1992) Rev. Invest. Clin. 44:215-220). P2 microglobulin isalso useful in staging myeloma patients (Pasqualetti et al. (1991) Eur.J. Cancer 27:1123-1126).

Cytogenetic aberrations may also be used as markers to create apredictive profile of a hematological malignancy. For example,chromosome abnormalities are found in a large percentage of CLL patientsand are helpful in predicting the course of CLL. For example, a 17pdeletion is indicative of aggressive disease progression. In addition,CLL patients with a chromosome 17p deletion or mutation in p53, or both,are known to respond poorly to chemotherapeutics and rituximab. Allelicloss on chromosome 17p may be also be a useful prognostic marker incolorectal cancer, where patients with a 17p deletion are associatedwith an increased tendency of disease dissemination in colorectal cancer(Khine et al. (1994) Cancer 73:28-35).

Deletions of the long arm of chromosome 11 (11q) are one of the mostfrequent structural chromosome aberrations in various types oflymphoproliferative disorders. CLL patients with chromosome 11q deletionand possibly ATM mutations have a poor survival compared to patientswithout either this defect or the 17p deletion. Furthermore, an 11qdeletion is often accompanied by extensive lymph node involvement(Dohner et al. (1997) Blood 89:2516-2522). This deletion also identifiespatients who are at high risk for disease persistence after high-dosetherapy and autologous transplantation.

The ataxia telangiectasia mutated (ATA4) gene is a tumor suppressor genethat is involved in cell cycle arrest, apoptosis, and repair of DNAdouble-strand breaks. It is found on chromosome 11. ATMmutations areassociated with increased risk for breast cancer among women with afamily history of breast cancer (Chenevix-Trench et al. (2002) J. Natl.Cancer Inst. 94:205-215; Thorstenson et al. (2003) Cancer Res.63:3325-3333) and/or early-onset breast cancers (Izatt et al. (1999)Genes Chromosomes Cancer 26:286-294; Teraoka et al. (2001) Cancer92:479-487). There is also a high frequency of association ofrhabdomyosarcoma with ATM gene mutation/deletion (Zhang et al. (2003)Cancer Biol. Ther. 1:87-91).

Methods for detecting chromosomal abnormalities in a patient are wellknown in the art (see, for example, Cuneo et al. (1999) Blood93:1372-1380; Dohner et al. (1997) Blood 89:2516-2522). Methods tomeasure mutated proteins, such as ATM, are well known in the art (see,for example, Butch et al. (2004) Clin. Chem. 50: 2302-2308).

Thus, the biomarkers that are evaluated in the methods described hereininclude the cell survival and apoptotic proteins described supra, andproteins involved in hematological malignancy-related signalingpathways. Determining the expression or presence can be at the proteinor nucleic acid level. Thus, the biomarkers include these proteins andthe genes encoding these proteins. Where detection is at the proteinlevel, the biomarker protein comprises the full-length polypeptide orany detectable fragment thereof, and can include variants of theseprotein sequences. Similarly, where detection is at the nucleotidelevel, the biomarker nucleic acid includes DNA comprising thefull-length coding sequence, a fragment of the full-length codingsequence, variants of these sequences, for example naturally occurringvariants or splice-variants, or the complement of such a sequence.Biomarker nucleic acids also include RNA, for example, mRNA, comprisingthe full-length sequence encoding the biomarker protein of interest, afragment of the full-length RNA sequence of interest, or variants ofthese sequences. Biomarker proteins and biomarker nucleic acids alsoinclude variants of these sequences. By “fragment” is intended a portionof the polynucleotide or a portion of the amino acid sequence and henceprotein encoded thereby. Polynucleotides that are fragments of abiomarker nucleotide sequence generally comprise at least 10, 15, 20,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides,or up to the number of nucleotides present in a full-length biomarkerpolynucleotide disclosed herein. A fragment of a biomarkerpolynucleotide will generally encode at least 15, 25, 30, 50, 100, 150,200, or 250 contiguous amino acids, or up to the total number of aminoacids present in a full-length biomarker protein of the invention.“Variant” is intended to mean substantially similar sequences.Generally, variants of a particular biomarker of the invention will haveat least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothat biomarker as determined by sequence alignment programs known in theart.

As provided above, any method known in the art can be used in themethods for determining the expression or presence of biomarkerdescribed herein. Circulating levels of biomarkers in a blood sampleobtained from a candidate subject, can be measured, for example, byELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Westernblot, multiplexing technologies, or other similar methods. Cell surfaceexpression of biomarkers can be measured, for example, by flowcytometry, immunohistochemistry, Western Blot, immunoprecipitation,magnetic bead selection, and quantification of cells expressing eitherof these cell surface markers. Biomarker RNA expression levels could bemeasured by RT-PCR, Qt-PCR, microarray, Northern blot, or other similartechnologies.

As previously noted, determining the expression or presence of thebiomarker of interest at the protein or nucleotide level can beaccomplished using any detection method known to those of skill in theart. By “detecting expression” or “detecting the level of” is intendeddetermining the expression level or presence of a biomarker protein orgene in the biological sample. Thus, “detecting expression” encompassesinstances where a biomarker is determined not to be expressed, not to bedetectably expressed, expressed at a low level, expressed at a normallevel, or overexpressed.

In certain aspects of the method provided herein, the one or moresubpopulation of lymphocytes are isolated, detected or measured. Incertain embodiments, the one or more subpopulation of lymphocytes areisolated, detected or measured using immunophenotyping techniques. Inother embodiments, the one or more subpopulation of lymphocytes areisolated, detected or measured using fluorescence activated cell sorting(FACS) techniques.

In certain embodiments of the methods provided herein, the one or morebiomarkers comprises ZAP-70, CD5, t(14;18), CD38, β-2 microglobulin, p53mutational status, ATM mutational status, chromosome 17p deletion,chromosome 11q deletion, surface or cytoplasmic immunoglobulin, CD138,CD25, 6q deletion, CD19, CD20, CD22, CD11c, CD 103, chromosome 7qdeletion, VH mutational status, or a combination thereof.

In certain aspects, the methods described herein, the determining steprequires determining the expression or presence of a combination ofbiomarkers. In certain embodiment, the combination of biomarkers is CD19and CD5 or CD20 and CD5.

In certain aspects, the expression or presence of these variousbiomarkers and any clinically useful prognostic markers in a biologicalsample can be detected at the protein or nucleic acid level, using, forexample, immunohistochemistry techniques or nucleic acid-basedtechniques such as in situ hybridization and RT-PCR. In one embodiments,the expression or presence of one or more biomarkers is carried out by ameans for nucleic acid amplification, a means for nucleic acidsequencing, a means utilizing a nucleic acid microarray (DNA and RNA),or a means for in situ hybridization using specifically labeled probes.

In other embodiments, the determining the expression or presence of oneor more biomarkers is carried out through gel electrophoresis. In oneembodiment, the determination is carried out through transfer to amembrane and hybridization with a specific probe.

In other embodiments, the determining the expression or presence of oneor more biomarkers carried out by a diagnostic imaging technique.

In still other embodiments, the determining the expression or presenceof one or more biomarkers carried out by a detectable solid substrate.In one embodiment, the detectable solid substrate is paramagneticnanoparticles functionalized with antibodies.

In another aspect, provided herein are methods for detecting ormeasuring residual lymphoma following a course of treatment in order toguide continuing or discontinuing treatment or changing from onetherapeutic regimen to another comprising determining the expression orpresence of one or more biomarkers from one or more subpopulation oflymphocytes in a subject wherein the course of treatment is treatmentwith a Btk inhibitor.

Methods for detecting expression of the biomarkers described herein, andoptionally cytokine markers, within the test and control biologicalsamples comprise any methods that determine the quantity or the presenceof these markers either at the nucleic acid or protein level. Suchmethods are well known in the art and include but are not limited towestern blots, northern blots, ELISA, immunoprecipitation,immunofluorescence, flow cytometry, immunohistochemistry, nucleic acidhybridization techniques, nucleic acid reverse transcription methods,and nucleic acid amplification methods. In particular embodiments,expression of a biomarker is detected on a protein level using, forexample, antibodies that are directed against specific biomarkerproteins. These antibodies can be used in various methods such asWestern blot, ELISA, multiplexing technologies, immunoprecipitation, orimmunohistochemistry techniques. In some embodiments, detection ofcytokine markers is accomplished by electrochemiluminescence (ECL).

Any means for specifically identifying and quantifying a biomarker (forexample, biomarker, a biomarker of cell survival or proliferation, abiomarker of apoptosis, a biomarker of a Btk-mediated signaling pathway)in the biological sample of a candidate subject is contemplated. Thus,in some embodiments, expression level of a biomarker protein of interestin a biological sample is detected by means of a binding protein capableof interacting specifically with that biomarker protein or abiologically active variant thereof. Preferably, labeled antibodies,binding portions thereof, or other binding partners may be used. Theword “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody soas to generate a “labeled” antibody. The label may be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

The antibodies for detection of a biomarker protein may be monoclonal orpolyclonal in origin, or may be synthetically or recombinantly produced.The amount of complexed protein, for example, the amount of biomarkerprotein associated with the binding protein, for example, an antibodythat specifically binds to the biomarker protein, is determined usingstandard protein detection methodologies known to those of skill in theart. A detailed review of immunological assay design, theory andprotocols can be found in numerous texts in the art (see, for example,Ausubel et al., eds. (1995) Current Protocols in Molecular Biology)(Greene Publishing and Wiley-Interscience, NY)); Coligan et al., eds.(1994) Current Protocols in Immunology (John Wiley & Sons, Inc., NewYork, N.Y.).

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of the marker is readilydeterminable to one skilled in the art. These labeled antibodies may beused in immunoassays as well as in histological applications to detectthe presence of any biomarker or protein of interest. The labeledantibodies may be polyclonal or monoclonal. Further, the antibodies foruse in detecting a protein of interest may be labeled with a radioactiveatom, an enzyme, a chromophoric or fluorescent moiety, or a colorimetrictag as described elsewhere herein. The choice of tagging label also willdepend on the detection limitations desired. Enzyme assays (ELISAs)typically allow detection of a colored product formed by interaction ofthe enzyme-tagged complex with an enzyme substrate. Radionuclides thatcan serve as detectable labels include, for example, I-131, I-123,I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. Examplesof enzymes that can serve as detectable labels include, but are notlimited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoricmoieties include, but are not limited to, fluorescein and rhodamine. Theantibodies may be conjugated to these labels by methods known in theart. For example, enzymes and chromophoric molecules may be conjugatedto the antibodies by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Alternatively, conjugationmay occur through a ligand-receptor pair. Examples of suitableligand-receptor pairs are biotin-avidin or biotin-streptavidin, andantibody-antigen.

In certain embodiments, expression or presence of one or more biomarkersor other proteins of interest within a biological sample, for example, asample of bodily fluid, is determined by radioimmunoassays orenzyme-linked immunoassays (ELISAs), competitive binding enzyme-linkedimmunoassays, dot blot (see, for example, Promega Protocols andApplications Guide (2^(nd) ed.; Promega Corporation (1991), Western blot(see, for example, Sambrook et al. (1989) Molecular Cloning, ALaboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), chromatography, preferably high performanceliquid chromatography (HPLC), or other assays known in the art. Thus,the detection assays can involve steps such as, but not limited to,immunoblotting, immunodiffusion, immunoelectrophoresis, orimmunoprecipitation.

In certain other embodiments, the methods of the invention are usefulfor identifying and treating hematological malignancies, including thoselisted above, that are refractory to (i.e., resistant to, or have becomeresistant to) first-line oncotherapeutic treatments.

The expression or presence of one or more of the biomarkers describedherein may also be determined at the nucleic acid level. Nucleicacid-based techniques for assessing expression are well known in the artand include, for example, determining the level of biomarker mRNA in abiological sample. Many expression detection methods use isolated RNA.Any RNA isolation technique that does not select against the isolationof mRNA can be utilized for the purification of RNA (see, e.g., Ausubelet al., ed. (1987-1999) Current Protocols in Molecular Biology (JohnWiley & Sons, New York). Additionally, large numbers of tissue samplescan readily be processed using techniques well known to those of skillin the art, such as, for example, the single-step RNA isolation processdisclosed in U.S. Pat. No. 4,843,155.

Thus, in some embodiments, the detection of a biomarker or other proteinof interest is assayed at the nucleic acid level using nucleic acidprobes. The term “nucleic acid probe” refers to any molecule that iscapable of selectively binding to a specifically intended target nucleicacid molecule, for example, a nucleotide transcript. Probes can besynthesized by one of skill in the art, or derived from appropriatebiological preparations. Probes may be specifically designed to belabeled, for example, with a radioactive label, a fluorescent label, anenzyme, a chemiluminescent tag, a colorimetric tag, or other labels ortags that are discussed above or that are known in the art. Examples ofmolecules that can be utilized as probes include, but are not limitedto, RNA and DNA.

For example, isolated mRNA can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onemethod for the detection of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to the mRNAencoded by the gene being detected. The nucleic acid probe can be, forexample, a full-length cDNA, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to an mRNA or genomic DNA encoding a biomarker, biomarkerdescribed herein above. Hybridization of an mRNA with the probeindicates that the biomarker or other target protein of interest isbeing expressed.

In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan canreadily adapt known mRNA detection methods for use in detecting thelevel of mRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of a mRNA of interest ina sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, biomarker expression isassessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System).

Expression levels of an RNA of interest may be monitored using amembrane blot (such as used in hybridization analysis such as Northern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The detection of expression may alsocomprise using nucleic acid probes in solution.

In one embodiment of the invention, microarrays are used to determineexpression or presence of one or more biomarkers. Microarrays areparticularly well suited for this purpose because of the reproducibilitybetween different experiments. DNA microarrays provide one method forthe simultaneous measurement of the expression levels of large numbersof genes. Each array consists of a reproducible pattern of captureprobes attached to a solid support. Labeled RNA or DNA is hybridized tocomplementary probes on the array and then detected by laser scanning.Hybridization intensities for each probe on the array are determined andconverted to a quantitative value representing relative gene expressionlevels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135,6,033,860, and 6,344,316, which are incorporated herein by reference.High-density oligonucleotide arrays are particularly useful fordetermining the gene expression profile for a large number of RNA's in asample.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261, incorporatedherein by reference in its entirety. Although a planar array surface ispreferred, the array may be fabricated on a surface of virtually anyshape or even a multiplicity of surfaces. Arrays may be peptides ornucleic acids on beads, gels, polymeric surfaces, fibers such as fiberoptics, glass or any other appropriate substrate, see U.S. Pat. Nos.5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of whichis hereby incorporated in its entirety for all purposes. Arrays may bepackaged in such a manner as to allow for diagnostics or othermanipulation of an all-inclusive device. See, for example, U.S. Pat.Nos. 5,856,174 and 5,922,591, herein incorporated by reference.

Pharmaceutical Compositions/Formulations

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. A summary of pharmaceuticalcompositions described herein may be found, for example, in Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated byreference in their entirety.

A pharmaceutical composition, as used herein, refers to a mixture of acompound described herein, such as, for example, compounds of Formula Dor the second agent, with other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients. The pharmaceutical composition facilitatesadministration of the compound to an organism. In practicing the methodsof treatment or use provided herein, therapeutically effective amountsof compounds described herein are administered in a pharmaceuticalcomposition to a mammal having a disease, disorder, or condition to betreated. Preferably, the mammal is a human. A therapeutically effectiveamount can vary widely depending on the severity of the disease, the ageand relative health of the subject, the potency of the compound used andother factors. The compounds can be used singly or in combination withone or more therapeutic agents as components of mixtures.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions may also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, areadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific intervening time limits,wherein such administration provides effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

The pharmaceutical formulations described herein can be administered toa subject by multiple administration routes, including but not limitedto, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular),intranasal, buccal, topical, rectal, or transdermal administrationroutes. The pharmaceutical formulations described herein include, butare not limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediateand controlled release formulations.

Pharmaceutical compositions including a compound described herein may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipientsin pharmaceutics and should be selected on the basis of compatibilitywith compounds disclosed herein, such as, compounds of any of Formula Dand the second agent, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

The term “disintegrate” includes both the dissolution and dispersion ofthe dosage form when contacted with gastrointestinal fluid.“Disintegration agents or disintegrants” facilitate the breakup ordisintegration of a substance. Examples of disintegration agents includea starch, e.g., a natural starch such as corn starch or potato starch, apregelatinized starch such as National 1551 or Amijel®, or sodium starchglycolate such as Promogel® or Explotab®, a cellulose such as a woodproduct, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101,Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, MingTia®, and Solka-Floc®, methylcellulose, croscarmellose, or across-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrosspovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

“Drug absorption” or “absorption” typically refers to the process ofmovement of drug from site of administration of a drug across a barrierinto a blood vessel or the site of action, e.g., a drug moving from thegastrointestinal tract into the portal vein or lymphatic system.

An “enteric coating” is a substance that remains substantially intact inthe stomach but dissolves and releases the drug in the small intestineor colon. Generally, the enteric coating comprises a polymeric materialthat prevents release in the low pH environment of the stomach but thationizes at a higher pH, typically a pH of 6 to 7, and thus dissolvessufficiently in the small intestine or colon to release the active agenttherein.

“Erosion facilitators” include materials that control the erosion of aparticular material in gastrointestinal fluid. Erosion facilitators aregenerally known to those of ordinary skill in the art. Exemplary erosionfacilitators include, e.g., hydrophilic polymers, electrolytes,proteins, peptides, and amino acids.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Flavoring agents” and/or “sweeteners” useful in the formulationsdescribed herein, include, e.g., acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate(MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mintcream, mixed berry, neohesperidine DC, neotame, orange, pear, peach,peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer,rum, saccharin, safrole, sorbitol, spearmint, spearmint cream,strawberry, strawberry cream, stevia, sucralose, sucrose, sodiumsaccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin,sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,wintergreen, xylitol, or any combination of these flavoring ingredients,e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon,chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus,orange-cream, vanilla-mint, and mixtures thereof.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

A “measurable serum concentration” or “measurable plasma concentration”describes the blood serum or blood plasma concentration, typicallymeasured in mg, □g, or ng of therapeutic agent per ml, dl, or l of bloodserum, absorbed into the bloodstream after administration. As usedherein, measurable plasma concentrations are typically measured in ng/mlor □g/ml.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at a site ofaction.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at a site ofaction.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Steady state,” as used herein, is when the amount of drug administeredis equal to the amount of drug eliminated within one dosing intervalresulting in a plateau or constant plasma drug exposure.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Dosage Forms

The compositions described herein can be formulated for administrationto a subject via any conventional means including, but not limited to,oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular),buccal, intranasal, rectal or transdermal administration routes. As usedherein, the term “subject” is used to mean an animal, preferably amammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

Moreover, the pharmaceutical compositions described herein, whichinclude a compound of any of Formula D or the second agent can beformulated into any suitable dosage form, including but not limited to,aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries,suspensions and the like, for oral ingestion by a patient to be treated,solid oral dosage forms, aerosols, controlled release formulations, fastmelt formulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include, forexample, fillers such as sugars, including lactose, sucrose, mannitol,or sorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In some embodiments, the solid dosage forms disclosed herein may be inthe form of a tablet, (including a suspension tablet, a fast-melttablet, a bite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder (including a sterilepackaged powder, a dispensable powder, or an effervescent powder) acapsule (including both soft or hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”),solid dispersion, solid solution, bioerodible dosage form, controlledrelease formulations, pulsatile release dosage forms, multiparticulatedosage forms, pellets, granules, or an aerosol. In other embodiments,the pharmaceutical formulation is in the form of a powder. In stillother embodiments, the pharmaceutical formulation is in the form of atablet, including but not limited to, a fast-melt tablet. Additionally,pharmaceutical formulations described herein may be administered as asingle capsule or in multiple capsule dosage form. In some embodiments,the pharmaceutical formulation is administered in two, or three, orfour, capsules or tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescenttablets, and capsules, are prepared by mixing particles of a compound ofany of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6), or Formula(D1-D6), with one or more pharmaceutical excipients to form a bulk blendcomposition. When referring to these bulk blend compositions ashomogeneous, it is meant that the particles of the compound of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), or Formula (D1-D6),are dispersed evenly throughout the composition so that the compositionmay be readily subdivided into equally effective unit dosage forms, suchas tablets, pills, and capsules. The individual unit dosages may alsoinclude film coatings, which disintegrate upon oral ingestion or uponcontact with diluent. These formulations can be manufactured byconventional pharmacological techniques.

Conventional pharmacological techniques include, e.g., one or acombination of methods: (1) dry mixing, (2) direct compression, (3)milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6)fusion. See, e.g., Lachman et al., The Theory and Practice of IndustrialPharmacy (1986). Other methods include, e.g., spray drying, pan coating,melt granulation, granulation, fluidized bed spray drying or coating(e.g., wurster coating), tangential coating, top spraying, tableting,extruding and the like.

The pharmaceutical solid dosage forms described herein can include acompound described herein and one or more pharmaceutically acceptableadditives such as a compatible carrier, binder, filling agent,suspending agent, flavoring agent, sweetening agent, disintegratingagent, dispersing agent, surfactant, lubricant, colorant, diluent,solubilizer, moistening agent, plasticizer, stabilizer, penetrationenhancer, wetting agent, anti-foaming agent, antioxidant, preservative,or one or more combination thereof. In still other aspects, usingstandard coating procedures, such as those described in Remington'sPharmaceutical Sciences, 20th Edition (2000), a film coating is providedaround the formulation of the compound of any of Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), or Formula (D1-D6). In one embodiment,some or all of the particles of the compound of any of Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), or Formula (D1-D6), are coated. Inanother embodiment, some or all of the particles of the compound of anyof Formula (A1-A6), Formula (B1-B6), Formula (C1-C6), or Formula(D1-D6), are microencapsulated. In still another embodiment, theparticles of the compound of any of Formula (A1-A6), Formula (B1-B6),Formula (C1-C6), or Formula (D1-D6), are not microencapsulated and areuncoated.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

In order to release the compound of any of Formula (A1-A6), Formula(B1-B6), Formula (C1-C6), or Formula (D1-D6), from a solid dosage formmatrix as efficiently as possible, disintegrants are often used in theformulation, especially when the dosage forms are compressed withbinder. Disintegrants help rupturing the dosage form matrix by swellingor capillary action when moisture is absorbed into the dosage form.Suitable disintegrants for use in the solid dosage forms describedherein include, but are not limited to, natural starch such as cornstarch or potato starch, a pregelatinized starch such as National 1551or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, acellulose such as a wood product, methylcrystalline cellulose, e.g.,Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100,Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose,croscarmellose, or a cross-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Formulators skilled in art can determine the binder level forthe formulations, but binder usage level of up to 70% in tabletformulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

The term “non water-soluble diluent” represents compounds typically usedin the formulation of pharmaceuticals, such as calcium phosphate,calcium sulfate, starches, modified starches and microcrystallinecellulose, and microcellulose (e.g., having a density of about 0.45g/cm³, e.g. Avicel, powdered cellulose), and talc.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described hereininclude, for example, e.g., butylated hydroxytoluene (BHT), sodiumascorbate, and tocopherol.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms described herein. The amounts of such additives can be readilydetermined by one skilled in the art, according to the particularproperties desired.

In other embodiments, one or more layers of the pharmaceuticalformulation are plasticized. Illustratively, a plasticizer is generallya high boiling point solid or liquid. Suitable plasticizers can be addedfrom about 0.01% to about 50% by weight (w/w) of the coatingcomposition. Plasticizers include, but are not limited to, diethylphthalate, citrate esters, polyethylene glycol, glycerol, acetylatedglycerides, triacetin, polypropylene glycol, polyethylene glycol,triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, andcastor oil.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend of the formulations described above. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating can provide a delayedrelease of the compound of any of Formula D or the second agent, fromthe formulation. In other embodiments, the film coating aids in patientcompliance (e.g., Opadry® coatings or sugar coating). Film coatingsincluding Opadry® typically range from about 1% to about 3% of thetablet weight. In other embodiments, the compressed tablets include oneor more excipients.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of the compound of any of Formula D or the second agent,described above, inside of a capsule. In some embodiments, theformulations (non-aqueous suspensions and solutions) are placed in asoft gelatin capsule. In other embodiments, the formulations are placedin standard gelatin capsules or non-gelatin capsules such as capsulescomprising HPMC. In other embodiments, the formulation is placed in asprinkle capsule, wherein the capsule may be swallowed whole or thecapsule may be opened and the contents sprinkled on food prior toeating. In some embodiments, the therapeutic dose is split into multiple(e.g., two, three, or four) capsules. In some embodiments, the entiredose of the formulation is delivered in a capsule form.

In various embodiments, the particles of the compound of any of FormulaD or the second agent, and one or more excipients are dry blended andcompressed into a mass, such as a tablet, having a hardness sufficientto provide a pharmaceutical composition that substantially disintegrateswithin less than about 30 minutes, less than about 35 minutes, less thanabout 40 minutes, less than about 45 minutes, less than about 50minutes, less than about 55 minutes, or less than about 60 minutes,after oral administration, thereby releasing the formulation into thegastrointestinal fluid.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein includematerials compatible with compounds of any of Formula D or the secondagent, which sufficiently isolate the compound of any of Formula D orthe second agent, from other non-compatible excipients. Materialscompatible with compounds of any of Formula D or the second agent, arethose that delay the release of the compounds of any of Formula D or thesecond agent, in vivo.

Exemplary microencapsulation materials useful for delaying the releaseof the formulations including compounds described herein, include, butare not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel®or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such asE461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

In still other embodiments, plasticizers such as polyethylene glycols,e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,stearic acid, propylene glycol, oleic acid, and triacetin areincorporated into the microencapsulation material. In other embodiments,the microencapsulating material useful for delaying the release of thepharmaceutical compositions is from the USP or the National Formulary(NF). In yet other embodiments, the microencapsulation material isKlucel. In still other embodiments, the microencapsulation material ismethocel.

Microencapsulated compounds of any of Formula D or the second agent, maybe formulated by methods known by one of ordinary skill in the art. Suchknown methods include, e.g., spray drying processes, spinningdisk-solvent processes, hot melt processes, spray chilling methods,fluidized bed, electrostatic deposition, centrifugal extrusion,rotational suspension separation, polymerization at liquid-gas orsolid-gas interface, pressure extrusion, or spraying solvent extractionbath. In addition to these, several chemical techniques, e.g., complexcoacervation, solvent evaporation, polymer-polymer incompatibility,interfacial polymerization in liquid media, in situ polymerization,in-liquid drying, and desolvation in liquid media could also be used.Furthermore, other methods such as roller compaction,extrusion/spheronization, coacervation, or nanoparticle coating may alsobe used.

In one embodiment, the particles of compounds of any of Formula D or thesecond agent, are microencapsulated prior to being formulated into oneof the above forms. In still another embodiment, some or most of theparticles are coated prior to being further formulated by using standardcoating procedures, such as those described in Remington'sPharmaceutical Sciences, 20th Edition (2000).

In other embodiments, the solid dosage formulations of the compounds ofany of Formula D or the second agent, are plasticized (coated) with oneor more layers. Illustratively, a plasticizer is generally a highboiling point solid or liquid. Suitable plasticizers can be added fromabout 0.01% to about 50% by weight (w/w) of the coating composition.Plasticizers include, but are not limited to, diethyl phthalate, citrateesters, polyethylene glycol, glycerol, acetylated glycerides, triacetin,polypropylene glycol, polyethylene glycol, triethyl citrate, dibutylsebacate, stearic acid, stearol, stearate, and castor oil.

In other embodiments, a powder including the formulations with acompound of any of Formula D or the second agent, described herein, maybe formulated to include one or more pharmaceutical excipients andflavors. Such a powder may be prepared, for example, by mixing theformulation and optional pharmaceutical excipients to form a bulk blendcomposition. Additional embodiments also include a suspending agentand/or a wetting agent. This bulk blend is uniformly subdivided intounit dosage packaging or multi-dosage packaging units.

In still other embodiments, effervescent powders are also prepared inaccordance with the present disclosure. Effervescent salts have beenused to disperse medicines in water for oral administration.Effervescent salts are granules or coarse powders containing a medicinalagent in a dry mixture, usually composed of sodium bicarbonate, citricacid and/or tartaric acid. When salts of the compositions describedherein are added to water, the acids and the base react to liberatecarbon dioxide gas, thereby causing “effervescence.” Examples ofeffervescent salts include, e.g., the following ingredients: sodiumbicarbonate or a mixture of sodium bicarbonate and sodium carbonate,citric acid and/or tartaric acid. Any acid-base combination that resultsin the liberation of carbon dioxide can be used in place of thecombination of sodium bicarbonate and citric and tartaric acids, as longas the ingredients were suitable for pharmaceutical use and result in apH of about 6.0 or higher.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. The enteric coated oral dosage form mayalso be a capsule (coated or uncoated) containing pellets, beads orgranules of the solid carrier or the composition, which are themselvescoated or uncoated.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above. It is expected that anyanionic polymer exhibiting a pH-dependent solubility profile can be usedas an enteric coating in the methods and compositions described hereinto achieve delivery to the lower gastrointestinal tract. In someembodiments the polymers described herein are anionic carboxylicpolymers. In other embodiments, the polymers and compatible mixturesthereof, and some of their properties, include, but are not limited to:

-   -   (a) Shellac, also called purified lac, a refined product        obtained from the resinous secretion of an insect. This coating        dissolves in media of pH >7;    -   (b) Acrylic polymers. The performance of acrylic polymers        (primarily their solubility in biological fluids) can vary based        on the degree and type of substitution. Examples of suitable        acrylic polymers include methacrylic acid copolymers and        ammonium methacrylate copolymers. The Eudragit series E, L, S,        RL, RS and NE (Rohm Pharma) are available as solubilized in        organic solvent, aqueous dispersion, or dry powders. The        Eudragit series RL, NE, and RS are insoluble in the        gastrointestinal tract but are permeable and are used primarily        for colonic targeting. The Eudragit series E dissolve in the        stomach. The Eudragit series L, L-30D and S are insoluble in        stomach and dissolve in the intestine;    -   (c) Cellulose Derivatives. Examples of suitable cellulose        derivatives are: ethyl cellulose; reaction mixtures of partial        acetate esters of cellulose with phthalic anhydride. The        performance can vary based on the degree and type of        substitution. Cellulose acetate phthalate (CAP) dissolves in        pH >6. Aquateric (FMC) is an aqueous based system and is a spray        dried CAP psuedolatex with particles <1 μm. Other components in        Aquateric can include pluronics, Tweens, and acetylated        monoglycerides. Other suitable cellulose derivatives include:        cellulose acetate trimellitate (Eastman); methylcellulose        (Pharmacoat, Methocel); hydroxypropylmethyl cellulose phthalate        (HPMCP); hydroxypropylmethyl cellulose succinate (HPMCS); and        hydroxypropylmethylcellulose acetate succinate (e.g., AQOAT        (Shin Etsu)). The performance can vary based on the degree and        type of substitution. For example, HPMCP such as, HP-50, HP-55,        HP-555, HP-55F grades are suitable. The performance can vary        based on the degree and type of substitution. For example,        suitable grades of hydroxypropylmethylcellulose acetate        succinate include, but are not limited to, AS-LG (LF), which        dissolves at pH 5, AS-MG (MF), which dissolves at pH 5.5, and        AS-HG (HF), which dissolves at higher pH. These polymers are        offered as granules, or as fine powders for aqueous dispersions;        Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves in pH >5,        and it is much less permeable to water vapor and gastric fluids.

In some embodiments, the coating can, and usually does, contain aplasticizer and possibly other coating excipients such as colorants,talc, and/or magnesium stearate, which are well known in the art.Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin(glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, anionic carboxylic acrylicpolymers usually will contain 10-25% by weight of a plasticizer,especially dibutyl phthalate, polyethylene glycol, triethyl citrate andtriacetin. Conventional coating techniques such as spray or pan coatingare employed to apply coatings. The coating thickness must be sufficientto ensure that the oral dosage form remains intact until the desiredsite of topical delivery in the intestinal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants(e.g., carnuba wax or PEG) may be added to the coatings besidesplasticizers to solubilize or disperse the coating material, and toimprove coating performance and the coated product.

In other embodiments, the formulations described herein, which includecompounds of Formula D or the second agent, are delivered using apulsatile dosage form. A pulsatile dosage form is capable of providingone or more immediate release pulses at predetermined time points aftera controlled lag time or at specific sites. Many other types ofcontrolled release systems known to those of ordinary skill in the artand are suitable for use with the formulations described herein.Examples of such delivery systems include, e.g., polymer-based systems,such as polylactic and polyglycolic acid, plyanhydrides andpolycaprolactone; porous matrices, nonpolymer-based systems that arelipids, including sterols, such as cholesterol, cholesterol esters andfatty acids, or neutral fats, such as mono-, di- and triglycerides;hydrogel release systems; silastic systems; peptide-based systems; waxcoatings, bioerodible dosage forms, compressed tablets usingconventional binders and the like. See, e.g., Liberman et al.,Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214 (1990); Singh etal., Encyclopedia of Pharmaceutical Technology, 2^(nd) Ed., pp. 751-753(2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509, 5,461,140,5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410, 5,977,175,6,465,014 and 6,932,983, each of which is specifically incorporated byreference.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of the compounds of any of Formula D or the secondagent, described herein and at least one dispersing agent or suspendingagent for oral administration to a subject. The formulations may be apowder and/or granules for suspension, and upon admixture with water, asubstantially uniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2^(nd) Ed., pp. 754-757(2002). In addition to the particles of compounds of Formula (A1-A6),the liquid dosage forms may include additives, such as: (a)disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) atleast one preservative, (e) viscosity enhancing agents, (f) at least onesweetening agent, and (g) at least one flavoring agent. In someembodiments, the aqueous dispersions can further include a crystallineinhibitor.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

Examples of disintegrating agents for use in the aqueous suspensions anddispersions include, but are not limited to, a starch, e.g., a naturalstarch such as corn starch or potato starch, a pregelatinized starchsuch as National 1551 or Amijel®, or sodium starch glycolate such asPromogel® or Explotab®; a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose; a cross-linked starch such as sodium starch glycolate; across-linked polymer such as crospovidone; a cross-linkedpolyvinylpyrrolidone; alginate such as alginic acid or a salt of alginicacid such as sodium alginate; a clay such as Veegum® HV (magnesiumaluminum silicate); a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth; sodium starch glycolate; bentonite; a naturalsponge; a surfactant; a resin such as a cation-exchange resin; citruspulp; sodium lauryl sulfate; sodium lauryl sulfate in combinationstarch; and the like.

In some embodiments, the dispersing agents suitable for the aqueoussuspensions and dispersions described herein are known in the art andinclude, for example, hydrophilic polymers, electrolytes, Tween® 60 or80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®),and the carbohydrate-based dispersing agents such as, for example,hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L), hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and HPMCK100M), carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,hydroxypropylmethyl-cellulose acetate stearate, noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer (Plasdone®,e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethyleneoxide and formaldehyde (also known as tyloxapol), poloxamers (e.g.,Pluronics F68®, F88®, and F108®, which are block copolymers of ethyleneoxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, alsoknown as Poloxamine 908®, which is a tetrafunctional block copolymerderived from sequential addition of propylene oxide and ethylene oxideto ethylenediamine (BASF Corporation, Parsippany, N.J.)). In otherembodiments, the dispersing agent is selected from a group notcomprising one of the following agents: hydrophilic polymers;electrolytes; Tween® 60 or 80; PEG; polyvinylpyrrolidone (PVP);hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M,and Pharmacoat® USP 2910 (Shin-Etsu)); carboxymethylcellulose sodium;methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl-cellulosephthalate; hydroxypropylmethyl-cellulose acetate stearate;non-crystalline cellulose; magnesium aluminum silicate; triethanolamine;polyvinyl alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics F68®,F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); or poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®).

Wetting agents suitable for the aqueous suspensions and dispersionsdescribed herein are known in the art and include, but are not limitedto, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens® such as e.g.,Tween 20® and Tween 80® (ICI Specialty Chemicals)), and polyethyleneglycols (e.g., Carbowaxs 3350® and 1450®, and Carbopol 934® (UnionCarbide)), oleic acid, glyceryl monostearate, sorbitan monooleate,sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodiumlauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodiumtaurocholate, simethicone, phosphotidylcholine and the like

Suitable preservatives for the aqueous suspensions or dispersionsdescribed herein include, for example, potassium sorbate, parabens(e.g., methylparaben and propylparaben), benzoic acid and its salts,other esters of parahydroxybenzoic acid such as butylparaben, alcoholssuch as ethyl alcohol or benzyl alcohol, phenolic compounds such asphenol, or quaternary compounds such as benzalkonium chloride.Preservatives, as used herein, are incorporated into the dosage form ata concentration sufficient to inhibit microbial growth.

Suitable viscosity enhancing agents for the aqueous suspensions ordispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, Plasdon® S-630, carbomer,polyvinyl alcohol, alginates, acacia, chitosans and combinationsthereof. The concentration of the viscosity enhancing agent will dependupon the agent selected and the viscosity desired.

Examples of sweetening agents suitable for the aqueous suspensions ordispersions described herein include, for example, acacia syrup,acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream,berry, black currant, butterscotch, calcium citrate, camphor, caramel,cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citruspunch, citrus cream, cotton candy, cocoa, cola, cool cherry, coolcitrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose,fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup,grape, grapefruit, honey, isomalt, lemon, lime, lemon cream,monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple,marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet®Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfamepotassium, mannitol, talin, sucralose, sorbitol, swiss cream, tagatose,tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wildcherry, wintergreen, xylitol, or any combination of these flavoringingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint,menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof. Inone embodiment, the aqueous liquid dispersion can comprise a sweeteningagent or flavoring agent in a concentration ranging from about 0.001% toabout 1.0% the volume of the aqueous dispersion. In another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.005% to about0.5% the volume of the aqueous dispersion. In yet another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.01% to about1.0% the volume of the aqueous dispersion.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

In some embodiments, the pharmaceutical formulations described hereincan be self-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion. SEDDS, as opposed to emulsions or microemulsions,spontaneously form emulsions when added to an excess of water withoutany external mechanical dispersion or agitation. An advantage of SEDDSis that only gentle mixing is required to distribute the dropletsthroughout the solution. Additionally, water or the aqueous phase can beadded just prior to administration, which ensures stability of anunstable or hydrophobic active ingredient. Thus, the SEDDS provides aneffective delivery system for oral and parenteral delivery ofhydrophobic active ingredients. SEDDS may provide improvements in thebioavailability of hydrophobic active ingredients. Methods of producingself-emulsifying dosage forms are known in the art and include, but arenot limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and6,960,563, each of which is specifically incorporated by reference.

It is to be appreciated that there is overlap between the above-listedadditives used in the aqueous dispersions or suspensions describedherein, since a given additive is often classified differently bydifferent practitioners in the field, or is commonly used for any ofseveral different functions. Thus, the above-listed additives should betaken as merely exemplary, and not limiting, of the types of additivesthat can be included in formulations described herein. The amounts ofsuch additives can be readily determined by one skilled in the art,according to the particular properties desired.

Intranasal Formulations

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated by reference. Formulations thatinclude a compound of any of Formula (A1-A6), Formula (B1-B6), Formula(C1-C6), or Formula (D1-D6), which are prepared according to these andother techniques well-known in the art are prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art. See, for example, Ansel, H. C. et al., Pharmaceutical DosageForms and Drug Delivery Systems, Sixth Ed. (1995). Preferably thesecompositions and formulations are prepared with suitable nontoxicpharmaceutically acceptable ingredients. These ingredients are known tothose skilled in the preparation of nasal dosage forms and some of thesecan be found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21stedition, 2005, a standard reference in the field. The choice of suitablecarriers is highly dependent upon the exact nature of the nasal dosageform desired, e.g., solutions, suspensions, ointments, or gels. Nasaldosage forms generally contain large amounts of water in addition to theactive ingredient. Minor amounts of other ingredients such as pHadjusters, emulsifiers or dispersing agents, preservatives, surfactants,gelling agents, or buffering and other stabilizing and solubilizingagents may also be present. The nasal dosage form should be isotonicwith nasal secretions.

For administration by inhalation, the compounds of any of Formula D orthe second agent, described herein may be in a form as an aerosol, amist or a powder. Pharmaceutical compositions described herein areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, such as, by way of example only, gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compounddescribed herein and a suitable powder base such as lactose or starch.

Buccal Formulations

Buccal formulations that include compounds of any of Formula D or thesecond agent may be administered using a variety of formulations knownin the art. For example, such formulations include, but are not limitedto, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136, eachof which is specifically incorporated by reference. In addition, thebuccal dosage forms described herein can further include a bioerodible(hydrolysable) polymeric carrier that also serves to adhere the dosageform to the buccal mucosa. The buccal dosage form is fabricated so as toerode gradually over a predetermined time period, wherein the deliveryof the compound of any of Formula D or the second agent, is providedessentially throughout. Buccal drug delivery, as will be appreciated bythose skilled in the art, avoids the disadvantages encountered with oraldrug administration, e.g., slow absorption, degradation of the activeagent by fluids present in the gastrointestinal tract and/or first-passinactivation in the liver. With regard to the bioerodible (hydrolysable)polymeric carrier, it will be appreciated that virtually any suchcarrier can be used, so long as the desired drug release profile is notcompromised, and the carrier is compatible with the compound of any ofFormula D or the second agent, and any other components that may bepresent in the buccal dosage unit. Generally, the polymeric carriercomprises hydrophilic (water-soluble and water-swellable) polymers thatadhere to the wet surface of the buccal mucosa. Examples of polymericcarriers useful herein include acrylic acid polymers and co, e.g., thoseknown as “carbomers” (Carbopol®, which may be obtained from B.F.Goodrich, is one such polymer). Other components may also beincorporated into the buccal dosage forms described herein include, butare not limited to, disintegrants, diluents, binders, lubricants,flavoring, colorants, preservatives, and the like. For buccal orsublingual administration, the compositions may take the form oftablets, lozenges, or gels formulated in a conventional manner.

Transdermal Formulations

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144, each of which is specifically incorporated by reference inits entirety.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiments, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound ofany of Formula D or the second agent; (2) a penetration enhancer; and(3) an aqueous adjuvant. In addition, transdermal formulations caninclude additional components such as, but not limited to, gellingagents, creams and ointment bases, and the like. In some embodiments,the transdermal formulation can further include a woven or non-wovenbacking material to enhance absorption and prevent the removal of thetransdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein can maintain a saturated orsupersaturated state to promote diffusion into the skin.

Formulations suitable for transdermal administration of compoundsdescribed herein may employ transdermal delivery devices and transdermaldelivery patches and can be lipophilic emulsions or buffered, aqueoussolutions, dissolved and/or dispersed in a polymer or an adhesive. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents. Still further, transdermal deliveryof the compounds described herein can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery of the compounds of any of Formula D orthe second agent. The rate of absorption can be slowed by usingrate-controlling membranes or by trapping the compound within a polymermatrix or gel. Conversely, absorption enhancers can be used to increaseabsorption. An absorption enhancer or carrier can include absorbablepharmaceutically acceptable solvents to assist passage through the skin.For example, transdermal devices are in the form of a bandage comprisinga backing member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundto the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.

Injectable Formulations

Formulations that include a compound of any of Formula D or the secondagent, suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Other Formulations

In certain embodiments, delivery systems for pharmaceutical compoundsmay be employed, such as, for example, liposomes and emulsions. Incertain embodiments, compositions provided herein can also include anmucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administeredtopically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compounds can contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

The compounds described herein may also be formulated in rectalcompositions such as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas, containingconventional suppository bases such as cocoa butter or other glycerides,as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and thelike. In suppository forms of the compositions, a low-melting wax suchas, but not limited to, a mixture of fatty acid glycerides, optionallyin combination with cocoa butter is first melted.

Dosing and Treatment Regimens

Disclosed herein, in certain embodiments, is a method for treating ahematological malignancy in an individual in need thereof, comprising:(a) administering to the individual an amount of an irreversible Btkinhibitor sufficient to mobilize a plurality of cells from themalignancy; and (b) analyzing the mobilized plurality of cells. In someembodiments, the amount of the irreversible Btk inhibitor is sufficientto induce lymphocytosis of a plurality of cells from the malignancy. Insome embodiments, the amount of the irreversible Btk inhibitor is from300 mg/day up to, and including, 1000 mg/day. In some embodiments, theamount of the irreversible Btk inhibitor is from 420 mg/day up to, andincluding, 840 mg/day. In some embodiments, the amount of theirreversible Btk inhibitor is about 420 mg/day, about 560 mg/day, orabout 840 mg/day. In some embodiments, the amount of the irreversibleBtk inhibitor is about 420 mg/day. In some embodiments, the AUC₀₋₂₄ ofthe Btk inhibitor is between about 150 and about 3500 ng*h/mL. In someembodiments, the AUC₀₋₂₄ of the Btk inhibitor is between about 500 andabout 1100 ng*h/mL. In some embodiments, the Btk inhibitor isadministered orally. In some embodiments, the Btk inhibitor isadministered once per day, twice per day, or three times per day. Insome embodiments, the Btk inhibitor is administered until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered daily until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered every other day untildisease progression, unacceptable toxicity, or individual choice. Insome embodiments, the Btk inhibitor is a maintenance therapy.

The compounds described herein can be used in the preparation ofmedicaments for the inhibition of Btk or a homolog thereof, or for thetreatment of diseases or conditions that would benefit, at least inpart, from inhibition of Btk or a homolog thereof, including a patientand/or subject diagnosed with a hematological malignancy. In addition, amethod for treating any of the diseases or conditions described hereinin a subject in need of such treatment, involves administration ofpharmaceutical compositions containing at least one compound of any ofFormula (A), Formula (B), Formula (C), or Formula (D), described herein,or a pharmaceutically acceptable salt, pharmaceutically acceptableN-oxide, pharmaceutically active metabolite, pharmaceutically acceptableprodrug, or pharmaceutically acceptable solvate thereof, intherapeutically effective amounts to said subject.

The compositions containing the compound(s) described herein can beadministered for prophylactic, therapeutic, or maintenance treatment. Insome embodiments, compositions containing the compounds described hereinare administered for therapeutic applications (e.g., administered to apatient diagnosed with a hematological malignancy). In some embodiments,compositions containing the compounds described herein are administeredfor therapeutic applications (e.g., administered to a patientsusceptible to or otherwise at risk of developing a hematologicalmalignancy). In some embodiments, compositions containing the compoundsdescribed herein are administered to a patient who is in remission as amaintenance therapy.

Amounts of a compound disclosed herein will depend on the use (e.g.,therapeutic, prophylactic, or maintenance). Amounts of a compounddisclosed herein will depend on severity and course of the disease orcondition, previous therapy, the patient's health status, weight, andresponse to the drugs, and the judgment of the treating physician. It isconsidered well within the skill of the art for one to determine suchtherapeutically effective amounts by routine experimentation (including,but not limited to, a dose escalation clinical trial). In someembodiments, the amount of the irreversible Btk inhibitor is from 300mg/day up to, and including, 1000 mg/day. In some embodiments, theamount of the irreversible Btk inhibitor is from 420 mg/day up to, andincluding, 840 mg/day. In some embodiments, the amount of the Btkinhibitor is from 400 mg/day up to, and including, 860 mg/day. In someembodiments, the amount of the Btk inhibitor is about 360 mg/day. Insome embodiments, the amount of the Btk inhibitor is about 420 mg/day.In some embodiments, the amount of the Btk inhibitor is about 560mg/day. In some embodiments, the amount of the Btk inhibitor is about840 mg/day. In some embodiments, the amount of the Btk inhibitor is from2 mg/kg/day up to, and including, 13 mg/kg/day. In some embodiments, theamount of the Btk inhibitor is from 2.5 mg/kg/day up to, and including,8 mg/kg/day. In some embodiments, the amount of the Btk inhibitor isfrom 2.5 mg/kg/day up to, and including, 6 mg/kg/day. In someembodiments, the amount of the Btk inhibitor is from 2.5 mg/kg/day upto, and including, 4 mg/kg/day. In some embodiments, the amount of theBtk inhibitor is about 2.5 mg/kg/day. In some embodiments, the amount ofthe Btk inhibitor is about 8 mg/kg/day.

In some embodiments, a Btk inhibitor disclosed herein is administereddaily. In some embodiments, a Btk inhibitor disclosed herein isadministered every other day.

In some embodiments, a Btk inhibitor disclosed herein is administeredonce per day. In some embodiments, a Btk inhibitor disclosed herein isadministered twice per day. In some embodiments, a Btk inhibitordisclosed herein is administered here times per day. In someembodiments, a Btk inhibitor disclosed herein is administered times perper day.

In some embodiments, the Btk inhibitor is administered until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered daily until diseaseprogression, unacceptable toxicity, or individual choice. In someembodiments, the Btk inhibitor is administered every other day untildisease progression, unacceptable toxicity, or individual choice.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, theseverity of the disease, the identity (e.g., weight) of the subject orhost in need of treatment, but can nevertheless be routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In general, however, doses employed for adult human treatmentwill typically be in the range of 0.02-5000 mg per day, or from about1-1500 mg per day. The desired dose may conveniently be presented in asingle dose or as divided doses administered simultaneously (or over ashort period of time) or at appropriate intervals, for example as two,three, four or more sub-doses per day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative. In some embodiments,each unit dosage form comprises 210 mg of a compound disclosed herein.In some embodiments, an individual is administered 1 unit dosage formper day. In some embodiments, an individual is administered 2 unitdosage forms per day. In some embodiments, an individual is administered3 unit dosage forms per day. In some embodiments, an individual isadministered 4 unit dosage forms per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD₅₀ and ED₅₀. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Kits/Articles of Manufacture

The present invention also encompasses kits for carrying out the methodsof the present invention. For example, the kit can comprise a labeledcompound or agent capable of detecting a biomarker described herein,e.g., a biomarker of apoptosis, cellular proliferation or survival, or aBtk-mediated signaling pathway, either at the protein or nucleic acidlevel, in a biological sample and means for determining the amount ofthe biomarker in the sample (for example, an antibody or anoligonucleotide probe that binds to RNA encoding a biomarker ofinterest) following incubation of the sample with a BCLD therapeuticagent of interest. Kits can be packaged to allow for detection ofmultiple biomarkers of interest by including individual labeledcompounds or agents capable of detecting each individual biomarker ofinterest and means for determining the amount of each biomarker in thesample.

The particular choice of the second agent used will depend upon thediagnosis of the attending physicians and their judgment of thecondition of the patient and the appropriate treatment protocol of theBtk inhibitors.

EXAMPLES

The following specific and non-limiting examples are to be construed asmerely illustrative, and do not limit the present disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentdisclosure to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety. Where reference ismade to a URL or other such identifier or address, it is understood thatsuch identifiers can change and particular information on the internetcan come and go, but equivalent information can be found by searchingthe internet. Reference thereto evidences the availability and publicdissemination of such information.

Example 1: Treatment of Non-Hodgkin Lymphoma by Administering a BtkInhibitor to Induce Pharmaceutical Debulking

Two groups of patients with Non-Hodgkin Lymphoma (15 each) are treatedwith or without a Btk inhibitor followed by administering a second agent(Taxane). Group 1 is subject to the second agent treatment only (Taxane)and Group 2 is subject to a Btk inhibitor treatment for 2 days followedby administering the second agent based on the determined expression orpresence of one or more B-cell lymphoproliferative disorder (BCLD)biomarkers from one or more subpopulation of lymphocytes.

Example 2. Determining the Expression or Presence of BCLD afterAdministering the Btk Inhibitor for the Treatment of Non-HodgkinLymphoma

Determining the expression or presence of BCLD after administeringcompound 15 to a patient of Group 1 is used by the known procedures.

Example 3. Use of Taxane for the Treatment of Non-Hodgkin Lymphoma

Following determination of the expression or presence of one or moreB-cell lymphoproliferative disorder (BCLD) biomarkers from one or moresubpopulation of lymphocytes in the patient, Taxane is used for Group 2patients.

Example 4: Clinical Example of Determination of BCLDs Using a BtkInhibitor

A patient with BCLD completes treatment with a Btk inhibitor or anothertreatment, and appears to be in complete remission. After this treatmentis stopped, a short course of the Btk inhibitor is then given. If cellswith markers of the malignant cells appear in the peripheral blood, insome embodiments it is an indication for continued treatment or forstarting another treatment. One example of the cell subpopulationinvestigated for in the peripheral blood is cells bearing both the CD5and CD20 markers, which is typical of CLL/SLL and Mantle Cell Lymphoma.These markers can be detectable by flow cytometry. A further example ofcell type is follicular lymphoma, which is characterized by cells witht(14;18) which in other embodiments are detectable by PCR or in situhybridization in cells harvested from the peripheral blood.

Based on the markers of the malignant cells as determined in theperipheral blood, a suitable second treatment regimen is administered.

Example 5: Pharmaceutical Compositions

The compositions described below are presented with a compound ofFormula (D) for illustrative purposes; any of the compounds of any ofFormulas (A), (B), (C), or (D) can be used in such pharmaceuticalcompositions.

Example 5a: Parenteral Composition

To prepare a parenteral pharmaceutical composition suitable foradministration by injection, 100 mg of a water-soluble salt of acompound of Formula (D) is dissolved in DMSO and then mixed with 10 mLof 0.9% sterile saline. The mixture is incorporated into a dosage unitform suitable for administration by injection.

Example 5b: Oral Composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of acompound of Formula (D) is mixed with 750 mg of starch. The mixture isincorporated into an oral dosage unit for, such as a hard gelatincapsule, which is suitable for oral administration.

Example 5c: Sublingual (Hard Lozenge) Composition

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound of Formula (D), with 420 mg ofpowdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mL distilledwater, and 0.42 mL mint extract. The mixture is gently blended andpoured into a mold to form a lozenge suitable for buccal administration.

Example 5d: Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound of Formula (D) is mixed with 50 mg of anhydrous citricacid and 100 mL of 0.9% sodium chloride solution. The mixture isincorporated into an inhalation delivery unit, such as a nebulizer,which is suitable for inhalation administration.

Example 5e: Rectal Gel Composition

To prepare a pharmaceutical composition for rectal delivery, 100 mg of acompound of Formula (D) is mixed with 2.5 g of methylcellulose (1500mPa), 100 mg of methylparapen, 5 g of glycerin and 100 mL of purifiedwater. The resulting gel mixture is then incorporated into rectaldelivery units, such as syringes, which are suitable for rectaladministration.

Example 5f: Topical Gel Composition

To prepare a pharmaceutical topical gel composition, 100 mg of acompound of Formula (D) is mixed with 1.75 g of hydroxypropyl cellulose,10 mL of propylene glycol, 10 mL of isopropyl myristate and 100 mL ofpurified alcohol USP. The resulting gel mixture is then incorporatedinto containers, such as tubes, which are suitable for topicaladministration.

Example 5g: Ophthalmic Solution Composition

To prepare a pharmaceutical ophthalmic solution composition, 100 mg of acompound of Formula (D) is mixed with 0.9 g of NaCl in 100 mL ofpurified water and filtered using a 0.2 micron filter. The resultingisotonic solution is then incorporated into ophthalmic delivery units,such as eye drop containers, which are suitable for ophthalmicadministration.

Example 6—Clinical Trial to Determine Efficacy of a Btk IrreversibleInhibitor in CLL and SLL Patients

Patients with CLL and/or SLL:

The data provided herein is a pooled analysis of patients with CLL orSLL from two clinical trials of a Btk irreversible inhibitor. Theinitial trial (Study 04753) was a Phase 1A multi-cohort, first-in-human,dose escalation trial of a Btk irreversible inhibitor in patients withrelapsed or refractory B-cell. 56 patients were enrolled between March2009 and September 2010 and two doses were evaluated, namely oralonce-daily dosing of a Btk irreversible inhibitor with a 28-day-on,7-day-off schedule, and a continuous daily oral dosing schedule. Of the56 patients enrolled, 16 CLL/SLL patients are included in this pooledanalysis.

The second trial (Study 1102) is a Phase 1B/II trial of two once-dailyoral doses of a Btk irreversible inhibitor in 2 populations of patientswith CLL or SLL; a cohort containing patients with relapsed ofrefractory disease after at least 2 prior treatment regimens, and asecond cohort of elderly patients with treatment-naïve disease. Thisstudy began enrollment in May 2010, and has enrolled 56 patients todate. For the purpose of this pooled analysis, 38 patients, with aminimum of 28 days follow-up and 28 patients with on study tumorassessments are included in this analysis. In sum, 56 patients from thetwo studies are included in this analysis.

The baseline characteristics of patients enrolled to the two studies aresummarized here. In study 04753, the median age was 66, there were 11patients with CLL and 5 patients with SLL. The median # of priortherapies was 3, with a range of 1-10. x % of patients had receivedprior nucleoside analogues, and x % had received prior anti-CD20 agents.

In study 1102, the median age was 68, 32 patients had CLL and 2 patientshad SLL. Of the patients with CLL, 10 had del 17p. 15 patients had bulkydisease, defined as a nodal mass >5 cm diameter. In therelapsed/refractory cohort, the median # of prior regimens was x.3 Perthe eligibility requirements, all patients had received a nucleosideanalogue-based regimen. 93% had received prior anti-CD20 agents, 9%alemtuzumab, and 19% bendamustine.

Objectives of the Analysis

The objective of this pooled analysis is to characterize the nature andkinetics of the response to a Btk irreversible inhibitor in CLL. The Btkirreversible inhibitor compound is one of a new class of BCR signalinginhibitors, and, similar to other inhibitors of this pathway, thekinetics of response differ between the peripheral blood and the nodalcompartments. The second objective was to summarize the current statusof the two studies with respect to best response, patient disposition,and time on treatment. The final objective was the summarization of theadverse event profile of the Btk inhibitor on a larger and more diversepopulation of patients with CLL or SLL.

Response Criteria

Different response criteria were applied to patients with CLL and SLLrespectively in these trials. Though considered biologically similar (oridentical) diseases, given the phenotypic differences in presentation,the IW criteria for CLL are based on improvement in circulatinglymphocytes, nodal/splenic/marrow-based disease, and normalization ofhematologic parameters. In contrast, the NHL criteria used to gauge thelymphomatous presentation of this disease (or SLL) are based only onimprovement in lymphadenopathy and organomegaly.

Lymphocyte Count

FIG. 5 depicts the change with treatment in the lymphocyte count for a57 year-old patient with disease relapse following multiple priortherapies and the poor-risk cytogenetic feature dell 1q began treatmentwith a Btk irreversible inhibitor nearly 6 months ago. Typical of themajority of CLL patients treated with a Btk irreversible inhibitor,there was an initial, rapid, and prominent reduction in nodal diseaseand spleen size, with a corresponding rise in the circulating lymphocytecount, likely a consequence of the inhibitory effects of a Btkirreversible inhibitor on lymphocyte homing to the nodal and spleniccompartments. Simultaneous with these changes, patients reportedsymptomatic improvement consistent with the resolution of bulky disease.Over time, the initial rise in lymphocytes returns to pre-treatmentlevels in spite of sustained reductions in adenopathy and splenomegaly.Cases such as this seen with a Btk irreversible inhibitor and similaragents, highlights the difficulty in applying standard response criteriato newer agents.

Effect of Treatment on Lymph Node SPD

As shown in FIG. 6, patients treated with a Btk irreversible inhibitorhad an immediate and marked nodal response to treatment. 85% ofevaluable patients achieved a partial response and even more had some LNshrinkage. 80% of patients with measurable LN disease achieved a 50%reduction in their SPD within 2 cycles of therapy. FIG. 7 shows theremarkable shrinkage in Lymph node post-treatment for the 57 year-oldpatient described supra.

Change in Lymph Node and Absolute Lymphocyte Count (ALC)

FIG. 8 depicts the effect of a Btk irreversible inhibitor on LN diseaseburden and lymphocytosis over time in the patients from the Phase Iatrial. Summary statistics from the patients with an early lymphocytosisshow a similar pattern in the median percent change over time in bothALC and in LN disease burden measured by the SPD. Immediately followingtreatment, patients develop an early lymphocytosis which decreases withtime to pre-treatment or normal levels. There is a sustained decrease indisease burden shown by the LN sum of perpendicular diameters. Thus,with some variability in timing, many patients show a marked decrease intumor burden in both peripheral blood and in LN disease with sustainedtreatment.

Adverse Effects

Adverse effects seen as a side effect of the treatment were monitored asoutlined in FIG. 9. The effects were categorized by severity into grades1-4. Grade 3 or greater events have been very uncommon. The vastmajority of events have been mild in severity. Diarrhea, nausea, andfatigue have been the most commonly reported adverse events, with mostof the reports occurring early in treatment

Thus, the oral Btk inhibitor has marked activity in patients with CLLand SLL including high-risk pts. It provides good disease control withlonger follow-up commonly exceeds 6 months. There is no evidence ofdrug-related myelosuppression or cumulative toxicity.

Example 7 Clinical Trial to Determine Safety and Efficacy of Compoundsof Formula (D)

The purpose of this clinical trial is to study the side effects and bestdose of a compound of Formula (D) and to determine its efficacy in thetreatment of patients diagnosed with recurrent B-cell lymphoma.

Study Design

Cohorts of 6 patients each receive a compound of Formula (D) at 1.25,2.5, 5.0, 8.3, 12.5, 17.5 mg/kg/d until the MTD is established. In caseswhere MTD is not reached, dosing levels are increased beyond 17.5mg/kg/d by 33% increments. Patients receive daily treatment for 28 daysfollowed by a 7 day rest period (one cycle). Tests for Btk occupancy bythe drug (“occupancy”) are performed on Day 1, 2, 8, 15 and 29 duringCycle 1 and on Day 1 and 15 of Cycles 3, 5, 7, 9, and 11. If ≦1 DLT(“dose-limiting toxicity”) is observed in the cohort during Cycle 1,escalation to the next cohort will proceed. Patients are enrolled in thenext cohort if four of the six patients enrolled in the cohort completedCycle 1 without experiencing a DLT, while the remaining two patients arecompleting evaluation. If ≧2 DLTs are observed during Cycle 1, dosing atthat dose and higher is suspended and the MTD is established as theprevious cohort. Patients are allowed to continue dosing at the MTD. If≧2 DLTs are seen at the 5.0 mg/kg/d cohort an additional cohort of 6patients can be added at 3.75 mg/kg/d.

Upon determination of the MTD, a cohort of 6 patients is enrolled toreceive a compound of Formula (D) at the MTD or “preferred occupyingdose” continuously for 35 days with no rest period (one cycle).

Study Population

Up to 52 patients with recurrent surface immunoglobulin positive B cellnon-Hodgkin's lymphoma according to WHO classification (including smalllymphocytic lymphoma/chronic lymphocytic leukemia)

Study Objectives

1. Primary Objectives include:

A. Determine pharmacokinetics (PK) of an orally administered compound ofFormula (D).

B. Evaluate tumor response. Patients have screening (i.e., baseline)disease assessments within 30 days before beginning treatment. Patientsundergo follow-up disease assessments following specified dosing cycles.Patients without evidence of disease progression on treatment arefollowed for a maximum of 6 months off treatment for diseaseprogression. At screening, a computed tomography (CT) (with contrastunless contraindicated) and positron-emission tomography (PET) or CT/PETscan of the chest, abdomen, and pelvis are required. At other visits, aCT (with contrast unless contraindicated) scan of the chest, abdomen,and pelvis are obtained. A CT/PET or PET is required to confirm acomplete response. Bone marrow biopsy is optional. In patients known tohave positive bone marrow before treatment with study drug, a repeatbiopsy should be done to confirm a complete response followingtreatment. All patients are evaluated for response based onInternational Working Group Revised Response Criteria for MalignantLymphoma, Guidelines for the diagnosis and treatment of chroniclymphocytic leukemia 14, or Uniform Response Criteria in Waldenstrom'sMacroglobulinemia.

C. Measure pharmacodynamic (PD) parameters to include drug occupancy ofBtk, the target enzyme, and effect on biological markers of B cellfunction. Specifically, this study examines the pharmacodynamics (PD) ofthe drug in peripheral blood mononuclear cells (PBMCs) using two PDassays. The first PD assay measures occupancy of the Btk active site bythe drug using a specially designed fluorescent probe. The second PDassay measures inhibition of B cell activation by stimulating the PBMCsex vivo at the BCR with anti-IgM/IgG, and then assaying cell surfaceexpression of the activation marker CD69 by flow cytometry The PDbiomarkers are measured in vitro from a blood sample removed frompatients 4-6 hours following an oral dose of the drug. These assaysdetermine what drug levels are required to achieve maximal occupancy ofBtk and maximal inhibition of BCR signaling. When possible, similarstudies are conducted on circulating tumor cells isolated from blood ofpatients.

2. Secondary Objectives include:

A. To analyze tumor biopsy samples (when possible) for apoptoticbiomarker expression analysis.

Inclusion Criteria

To be eligible to participate in this study, a patient must meet thefollowing criteria:

-   -   Women and men ≧18 years of age    -   Body weight ≧40 kg    -   Recurrent surface immunoglobulin positive B cell non-Hodgkin's        lymphoma (NHL) according to WHO classification, including small        lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and        lymphoplasmacytic lymphoma, including Waldenstrom's        Macroglobulinemia (WM)    -   Measurable disease (for NHL, bidimensional disease ≧2 cm        diameter in at least one dimension, for CLL ≧5000 leukemia        cells/mm3, and for WM presence of immunoglobulin M paraprotein        with a minimum IgM level ≧1000 mg/dL and infiltration of bone        marrow by lymphoplasmacytic cells)    -   Have failed ≧1 previous treatment for lymphoma and no standard        therapy is available. Patients with diffuse large B cell        lymphoma must have failed, refused or be ineligible for        autologous stem cell transplant    -   ECOG performance status of ≦1    -   Ability to swallow oral capsules without difficulty    -   Willing and able to sign a written informed consent

Exclusion Criteria

A patient meeting any of the following criteria will be excluded fromthis study:

-   -   More than four prior systemic therapies (not counting        maintenance rituximab), except for CLL patients. Salvage        therapy/conditioning regimen leading up to autologous bone        marrow transplantation is considered to be one regimen    -   Prior allogeneic bone marrow transplant    -   Immunotherapy, chemotherapy, radiotherapy or experimental        therapy within 4 weeks before first day of study drug dosing    -   Major surgery within 4 weeks before first day of study drug        dosing    -   CNS involvement by lymphoma    -   Active opportunistic infection or treatment for opportunistic        infection within 4 weeks before first day of study drug dosing    -   Uncontrolled illness including but not limited to: ongoing or        active infection, symptomatic congestive heart failure (New York        Heart Association Class III or IV heart failure), unstable        angina pectoris, cardiac arrhythmia, and psychiatric illness        that would limit compliance with study requirements    -   History of myocardial infarction, acute coronary syndromes        (including unstable angina), coronary angioplasty and/or        stenting within the past 6 months    -   Known HIV infection    -   Hepatitis B sAg or Hepatitis C positive    -   Other medical or psychiatric illness or organ dysfunction which,        in the opinion of the investigator, would either compromise the        patient's safety or interfere with the evaluation of the safety        of the study agent    -   Pregnant or lactating women (female patients of child-bearing        potential must have a negative serum pregnancy test within 14        days of first day of drug dosing, or, if positive, a pregnancy        ruled out by ultrasound)    -   History of prior cancer <2 years ago, except for basal cell or        squamous cell carcinoma of the skin, cervical cancer in situ or        other in situ carcinomas

Results:

29 pts (12 follicular, 7 CLL/SLL, 4 DLBCL, 4 mantle, 2 marginal) with amedian of 3 prior therapies have been enrolled on cohorts 1-4. Therapywas well tolerated with most adverse events <grade 2. One protocoldefined DLT (dose delay >7 d due to neutropenia) was observed. 19/22 ptsfrom cohorts 1-3 are evaluable. The ORR is 42%; 1 CR (SLL), 7 PR (4CLL/SLL, 2 MCL and 1FL). In cohort 2, PD demonstrate complete occupancyof Btk by a compound of Formula (D), with >95% enzyme occupancy 4 hourspost dose in all pts. Basophil degranulation, a Btk-dependent cellularprocess, was completely inhibited up to 24 hrs. T-cell responses werenot affected, and no significant depletion of peripheral blood B, T orNK cell counts was observed. Positive correlation (R2=0.93) was foundbetween Btk active-site occupancy in PBMCs (mean of Days 1 and 8) and acompound of Formula (D) plasma AUC0−° (Day 1) at the 1.25 mg/kg dose.

Example 8: Clinical Example of Diagnosis of BCLDs Using a Btk Inhibitor

A patient with BCLD completes treatment with a Btk inhibitor or anothertreatment, and appears to be in complete remission. After this treatmentis stopped, a short course of the Btk inhibitor is then given. If cellswith markers of the malignant cells appear in the peripheral blood, insome embodiments it is an indication for continued treatment or forstarting another treatment. One example of the cell subpopulationinvestigated for in the peripheral blood is cells bearing both the CD5and CD20 markers, which is typical of CLL/SLL and Mantle Cell Lymphoma.These markers can be detectable by flow cytometry. A further example ofcell type is follicular lymphoma, which is characterized by cells witht(14;18) which in other embodiments are detectable by PCR or in situhybridization in cells harvested from the peripheral blood.

For patients initially starting on treatment an increase of themalignant subpopulation can be an early predictive marker of response orduration of response.

For patients who have previously received treatment and are suspected ofprogressing based upon changes (for example in a scan) that arenon-diagnostic, the BTK test for peripheral blood cell increases couldadd diagnostic information that enable earlier treatment of relapse.This would be valuable in determining whether to re-start treatment forBCLD or to watch or to pursue an alternative diagnosis.

The test could yield better diagnostic information for patients whoseBCLD is suspected to be transforming into a more aggressive cellularform. For example both CLL/SLL and lower grade follicular lymphoma cantransform into a higher grade process which may resemble diffuse large Bcell lymphoma, and require more aggressive treatment.

Example 9: Patient Selection

Patient selection screens are performed to identify an individual withthe ABC subtype of DLBCL. Gene expression profiling is conducted usingFFPE biopsy material, using RNA amplified with a Nugen kit and assayedon an Affymetrix U133Plus 2.0 arrays.

Samples are screened for recurrent somatic mutations. This isaccomplished by conventional resequencing of candidate genes in theNF-kB and B cell receptor signaling pathways (e.g. CARD11, CD79A, CD79B,MYD88, TNFAIP3) plus p53 by exon amplification and standard dideoxyautomated DNA sequencing.

The patient selection screen also identifies patients with ABC DLBCLthat are particularly sensitive or resistant to Btk inhibitors. Apositive result for a CARD11 mutation indicates that the individual isresistant to Btk inhibitors because CARD11 mutations activate the NF-kBpathway at a step that is downstream of BTK.

Genomic copy number analysis is also required to adequately assess theactivity of oncogenic pathways that may be relevant for the response toBtk inhibitors as well as to assess prognosis. In particular, ABC DLBCLsharbor genomic deletions of the TNFAIP3 locus, which encodes A20, anegative regulator of NF-kB. Thus, a full assessment of A20 statusrequires both resequencing to look for somatic mutations and copy numberanalysis to look for deletions. In addition, patients are identifiedwith DLBCL tumors that harbor genomic deletions in the INK4a/ARF locusor have trisomy of chromosome 3 because these genomic aberrations areassociated with poor prognosis in ABC DLBCL. A single pass highthroughput DNA sequencing is performed using the Illumina HiSeq2000platform to assess genomic copy number globally.

Example 10: PK and Efficacy of a Btk Inhibitor in Individuals with CLLor SLL

A Btk inhibitor was administered to 33 individuals diagnosed with CLL orSLL. Efficacy and PK was determined.

Day 8 Dose AUC0-24 IWG Resp No. mg Patient_ID Group Sex (ng · h/mL)Cycle March 2011  1 420 073-203 Naïve Female 102 7 PR  2 420 217-107 R/RMale 120 8 PR  3 420 217-202 Naive Female 121 7 SD  4 420 032-110 R/RMale 155 6 PR  5 420 217-104 R/R Male 176 8 PR  6 420 032-201 Naïve Male177 9 PR  7 420 217-103 R/R Female 206 8 Nodal  8 420 032-104 R/R Male227 8 PR  9 420 217-102 R/R Male 243 9 Nodal 10 420 217-106 R/R Female267 8 Nodal 11 420 032-109 R/R Male 318 7 Nodal 12 420 217-110 R/RFemale 407 7 Nodal 13 420 038-101 R/R Male 428 7 PR 14 420 217-111 R/RMale 473 7 PR 15 420 217-109 R/R Male 498 7 Nodal 16 420 032-107 R/RMale 502 8 Nodal 17 420 073-201 Naïve Male 532 2 SD 18 420 032-105 R/RMale 534 8 PR 19 420 217-101 R/R Male 570 9 CR 20 420 073-101 R/R Male593 4 PR 21 420 217-105 R/R Female 594 8 PR 22 420 032-101 R/R Female643 9 Nodal 23 420 073-202 Naïve Male 648 9 PR 24 420 217-112 R/R Female653 7 SD 25 420 217-201 Naïve Male 687 9 PR 26 420 073-204 Naïve Male784 1 NE 27 420 217-108 R/R Male 809 1 PD 28 420 032-108 R/R Male 907 7PR 29 420 032-106 R/R Male 1200 8 Nodal 30 420 032-102 R/R Male 1210 2NE 31 420 217-113 R/R Male 1270 4 Cri 32 420 032-202 Naïve Female 1670 8PR 33 420 038-201 Naïve Female 2000 7 CR

Example 11: Clinical Trial with Btk Inhibitor

A phase Ib/II clinical trial was performed to study the effects of a Btkinhibitor on individuals with CLL.

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety Study

Intervention Model: Parallel Assignment

Masking: Open Label

Primary Purpose: Treatment

Group I (elderly, naïve, individuals) received 420 mg/day of the Btkinhibitor. Group II (R/R individuals, who had twice been treated withfludara) received 420 mg/day of the Btk inhibitor. Group III (R/Rindividuals, who had twice been treated with fludara) received 840mg/day of the Btk inhibitor.

Patient Characteristics Treatment- Relapsed/Re- Relapsed/Re- Naïvefractory fractory 420 mg 420 mg 840 mg (N = 23) (N = 27) (N = 33) Age, yMedian: 71 64 65 Range: 66-84 40-81 44-80 Dx, # pts CLL: 22 (96%) 26(96%) 32 (97%) SLL: 1 (4%) 1 (4%) 1 (3%) Prior Rx, # Median:  0  3  5Range:  2-10  2-12 Prior therapy, % Nucleoside analog 0 (0%) 27 (100%)33 (100%) Rituximab 0 (0%) 25 (93%) 32 (97%) Alkylator 0 (0%) 24 (89%)27 (82%) Alemtuzumab 0 (0%) 5 (19%) 3 (9%) Bendamustine 0 (0%) 8 (30%)13 (39%) Ofatumumab 0 (0%) 8 (30%) 10 (30%) Cytopenia at baseline, % ANC<1500/UL 1 (4%) 6 (22%) 17 (52%) HGB <11 g/dL 7 (30%) 4 (15%) 19 (58%)Platelets <100,000/uL 9 (39%) 8 (30%) 22 (67%) Prognostic Markers, %*IgVH unmutated: 8/16 (50%) 17/24 (71%) 18/24 (75%) Del(17p): 2/17 (12%)9/24 (38%) 10/25 (40%) Del(11q): 0/17 (0%) 8/24 (33%) 12/25 (48%) βMicroglobin <3 mg/L 10/16 (62%) 14/23 (61%) 8/25 (32%) β Microglobin ≧3mg/L 6/16 (38%) 9/23 (39%) 17/25 (68%)

Tumor assessment was performed every 2 treatment cycles.

Objectives

Describe the characteristics of the antitumor effect of a Btk inhibitorin individuals with CLL/SLL, e.g., reduction inlymphadenopathy/splenomegaly, and kinetics of change in absolutelymphocyte count (ACL).

Summarize the safety profile of the Btk inhibitor.

Inclusion Criteria

FOR TREATMENT-NAIVE GROUP ONLY: Men and women ≧65 years of age withconfirmed diagnosis of CLL/SLL, who require treatment per NCI orInternational Working Group guidelines 11-14

FOR RELAPSED/REFRACTORY GROUP ONLY: Men and women ≧18 years of age witha confirmed diagnosis of relapsed/refractory CLL/SLL unresponsive totherapy (ie, failed ≧2 previous treatments for CLL/SLL and at least 1regimen had to have had a purine analog [eg, fludarabine] for subjectswith CLL)

Body weight ≧40 kg

ECOG performance status of ≦2

Agreement to use contraception during the study and for 30 days afterthe last dose of study drug if sexually active and able to bear children

Willing and able to participate in all required evaluations andprocedures in this study protocol including swallowing capsules withoutdifficulty

Ability to understand the purpose and risks of the study and providesigned and dated informed consent and authorization to use protectedhealth information (in accordance with national and local subjectprivacy regulations)

Exclusion Criteria

A life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, interfere with the absorption or metabolism of Btkinhibitor PO, or put the study outcomes at undue risk

Any immunotherapy, chemotherapy, radiotherapy, or experimental therapywithin 4 weeks before first dose of study drug (corticosteroids fordisease-related symptoms allowed but require 1-week washout before studydrug administration)

Central nervous system (CNS) involvement by lymphoma

Major surgery within 4 weeks before first dose of study drug

Creatinine >1.5×institutional upper limit of normal (ULN); totalbilirubin >1.5×ULN (unless due to Gilbert's disease); and aspartateaminotransferase (AST) or alanine aminotransferase (ALT) >2.5×ULN unlessdisease related

Concomitant use of medicines known to cause QT prolongation or torsadesde pointes

Significant screening electrocardiogram (ECG) abnormalities includingleft bundle branch block, 2nd degree AV block type II, 3rd degree block,bradycardia, and QTc >470 msec

Lactating or pregnant

Response Criteria

NHL IWG criterial were applied to SLL cases without modification

The 2008 CLL IWG criteria were applied to CLL cases with the followingmodifications:

-   -   a. An isolated lymphocytosis, in the absence of other parameters        meeting the criteria for PD, was not considered PD    -   b. Patients experiencing a lymphocytosis, but obtaining a PR by        other measurable parameters, were classified as “nodal” response        until there was a 50% reduction in ALC from baseline in which        case they were categorized as PR.    -   c. Patients with a normal ALC (<5K) at baseline with        treatment-related lymphocytosis required normalization to <5K to        be categorized as PR.        Results

Subject Disposition Treatment- Relapsed/Re- Relapsed/Re- Naïve fractoryfractory 420 mg 420 mg 840 mg (N = 23) (N = 27) (N = 33) Number ofsubjects 23 27 33 Follow-up Median (months) 6.3 7.8 4.6 Range 1.4-9.20.7-9.5 0.3-6.5 Subjects still on study 21 (91%) 22 (81%) 28 (85%)Subject Discontinued 2 (9%)  5 (19%)  5 (15%) Primary ReasonsDiscontinuation Disease Progression 0 (0%) 2 (7%) 1 (3%) Death 0 (0%) 0(0%) 2 (6%) Adverse Event 1 (4%) 1 (4%) 1 (3%) Other 1 (4%) 2 (7%) 1(3%)

Best Response Treatment-Naïve Relapsed/Refractory 420 mg 420 mg N 21 27CR 1 (5%) 1 (4%) PR 13 (62%) 12 (44%) ORR % 67% 48% Nodal  4 (19%) 11(41%) SD  2 (10%) 1 (4%) PD  0 1 (4%) NE 1 (5%) 1 (4%)

Best Response by Risk Features Best Response Molecular Risk Feature NIWG Response Nodal Response Overall 27 48% 41% Del17p 9 44% 33% Del11q 863% 37% IgVH unmutated 17 53% 29%

Results further summarized in FIGS. 18-27. FIG. 18 presents theresponses for the naive, 420 mg/day group. FIG. 19 presents theresponses for the R/R, 420 mg/day group. FIG. 20 presents the responsesby prognostic factors. FIG. 21 presents responses over time. FIG. 22presents the best responses for all patients. FIG. 23 presents the bestresponses for abstract patients. FIG. 24 presents the best response byprognostic factor. FIG. 25 presents initial (Cycle 2) responseassessment and best response (420 mg Cohorts). FIG. 26 presents initial(Cycle 2) response assessment by dose: relapsed/refractory. FIG. 27presents improvements in hematological parameters.

Conclusions

The interim Phase II data confirm that a Btk inhibitor is highly activein both treatment-naïve and relapsed/refractory CLL/SLL patients

Class-specific rapid lymph node reduction with concurrent lymphocytosisseen in the majority of patients 2008 CLL IWG objective responses(PR+CR) and nodal responses appear to be durable and independent of highrisk genomic features

A high proportion (85%) of relapsed or refractory patients arefree-of-progression at 6 months (420 mg cohort)

Example 12: Long Term Follow-Up Trial for Individuals Taking BtkInhibitor

The purpose of this study is to determine the long-term safety of afixed-dose, daily regimen of Btk inhibitor PO in subjects with B celllymphoma or chronic lymphocytic leukemia/small lymphocytic leukemia(CLL/SLL).

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Intervention: 420 mg/day of a Btk inhibitor

Applicable conditions: B-cell Chronic Lymphocytic Leukemia; SmallLymphocytic Lymphoma; Diffuse Well-Differentiated Lymphocytic Lymphoma;B Cell Lymphoma; Follicular Lymphoma; Mantle Cell Lymphoma;Non-Hodgkin's Lymphoma; Waldenstrom Macroglobulinemia; Burkitt Lymphoma;B-Cell Diffuse Lymphoma

Primary Outcome Measures:

Adverse Events/Safety Tolerability [Time Frame: 30 days after last doseof study drug]—frequency, severity, and relatedness of adverse events

Secondary Outcome Measures:

Tumor Response [Time Frame: frequency of tumor assessments done perstandard of care]—tumor response will be assessed per establishedresponse criteria. This study will capture time to disease progressionand duration of response.

Tumor Response [Time Frame: Time to disease progression]—Duration ofresponse as measured by established response criteria for B celllymphoma and chronic lymphocytic leukemia

Inclusion Criteria

Men and women with B cell lymphoma or CLL/small lymphocytic lymphoma(SLL) who had stable disease or response to Btk inhibitor PO for atleast 6 months on a prior Btk inhibitor study and want to continue studydrug or who had disease progression on PCYC-04753 and want to try ahigher dose

Eastern Cooperative Oncology Group (ECOG) performance status of ≦2

Agreement to use contraception during the study and for 30 days afterthe last dose of study drug if sexually active and able to bear children

Willing and able to participate in all required evaluations andprocedures in this study protocol including swallowing capsules withoutdifficulty

Ability to understand the purpose and risks of the study and providesigned and dated informed consent and authorization to use protectedhealth information (in accordance with national and local subjectprivacy regulations)

Exclusion Criteria

A life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, interfere with the absorption or metabolism of Btkinhibitor PO, or put the study outcomes at undue risk

Concomitant immunotherapy, chemotherapy, radiotherapy, corticosteroids(at dosages equivalent to prednisone >20 mg/day), or experimentaltherapy

Concomitant use of medicines known to cause QT prolongation or torsadesde pointes

Central nervous system (CNS) involvement by lymphoma

Creatinine >1.5×institutional upper limit of normal (ULN); totalbilirubin >1.5×ULN (unless due to Gilbert's disease); and aspartateaminotransferase (AST) or alanine aminotransferase (ALT) >2.5×ULN unlessdisease related

Lactating or pregnant

Example 13: Phase II Study of Btk Inhibitor in R/R MCL

The purpose of this study is to: Evaluate the efficacy of Btk inhibitorin relapsed/refractory subjects with MCL who have not had priorbortezomib, and who have had prior bortezomib

The secondary objective is to evaluate the safety of a fixed dailydosing regimen of Btk inhibitor capsules in this population.

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Parallel Assignment

Masking: Open Label

Primary Purpose: Treatment

Intervention: 560 mg/day of a Btk inhibitor

Primary Outcome Measures

To Measure the Number of Participants with a Response to Study Drug[Time Frame: Participants will be followed until progression of diseaseor start of another anti-cancer treatment.]

Secondary Outcome Measures

To Measure the Number of Participants with Adverse Events as a Measureof Safety and Tolerability [Time Frame: Participants will be followeduntil progression of disease or start of another anti-cancer treatment.]

To Measure the Number of Participants Pharmacokinetics to Assist inDetermining How the Body Responds to the Study Drug [Time Frame:Procedure to be Performed During the First Month of Receiving StudyDrug.]

Patient Reported Outcomes [Time Frame: Participants will be followeduntil progression of disease or start of another anti-cancer treatment.]

To measure the number of participants reported outcomes in determiningthe health related quality of life.

Inclusion Criteria:

Men and women ≧18 years of age

ECOG performance status of ≦2

Pathologically confirmed MCL, with documentation of eitheroverexpression of cyclin D1 or t(11;14), and measurable disease on crosssectional imaging that is ≧2 cm in the longest diameter and measurablein 2 perpendicular dimensions

Documented failure to achieve at least partial response (PR) with, ordocumented disease progression disease after, the most recent treatmentregimen

At least 1, but no more than 5, prior treatment regimens for MCL (Note:Subjects having received ≧2 cycles of prior treatment with bortezomib,either as a single agent or as part of a combination therapy regimen,will be considered to be bortezomib-exposed.)

Willing and able to participate in all required evaluations andprocedures in this study protocol including swallowing capsules withoutdifficulty

Ability to understand the purpose and risks of the study and providesigned and dated informed consent and authorization to use protectedhealth information (in accordance with national and local subjectprivacy regulations)

Major Exclusion Criteria:

Prior chemotherapy within 3 weeks, nitrosoureas within 6 weeks,therapeutic anticancer antibodies within 4 weeks, radio- ortoxin-immunoconjugates within 10 weeks, radiation therapy within 3weeks, or major surgery within 2 weeks of first dose of study drug

Any life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, interfere with the absorption or metabolism of Btkinhibitor capsules, or put the study outcomes at undue risk

Clinically significant cardiovascular disease such as uncontrolled orsymptomatic arrhythmias, congestive heart failure, or myocardialinfarction within 6 months of screening, or any Class 3 or 4 cardiacdisease as defined by the New York Heart Association FunctionalClassification

Malabsorption syndrome, disease significantly affecting gastrointestinalfunction, or resection of the stomach or small bowel or ulcerativecolitis, symptomatic inflammatory bowel disease, or partial or completebowel obstruction

Any of the following laboratory abnormalities:

-   -   a. Absolute neutrophil count (ANC) <750 cells/mm3 (0.75×109/L)        unless there is documented bone marrow involvement    -   b. Platelet count <50,000 cells/mm3 (50×109/L) independent of        transfusion support unless there is documented bone marrow        involvement    -   c. Serum aspartate transaminase (AST/SGOT) or alanine        transaminase (ALT/SGPT) ≧3.0×upper limit of normal (ULN)    -   d. Creatinine >2.0×ULN

Example 14: Phase II Study of Btk Inhibitor+Ofatumumab in R/R CLL

The purpose of this study was to determine the efficacy and safety of afixed-dose, daily regimen of orally administered Btk inhibitor combinedwith ofatumumab in subjects with relapsed/refractory CLL/SLL and relateddiseases

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Intervention: 420 mg/day of a Btk inhibitor, standard dose of ofatumumab

Applicable conditions: B-cell Chronic Lymphocytic Leukemia; SmallLymphocytic Lymphoma; Diffuse Well-Differentiated Lymphocytic Lymphoma;Prolymphocytic Leukemia; Richter's Transformation

Primary Outcome Measures:

Response and safety of Btk inhibitor [Time Frame: At the end of cycles 1and 3]

Response rate as defined by recent guidelines in Chronic LymphocyticLeukemia

Secondary Outcome Measures:

Pharmacokinetic/Pharmacodynamic assessments [Time Frame: during 1-2cycles]

Pharmacodynamics of Btk inhibitor (ie, drug occupancy of Btk and effecton biological market 1/2) of Btk inhibitor.

Tumor Response [Time Frame: at the end of Cycles 2, 4 and 6 (28 days foreach cycle)]

Overall response rate as defined by recent guidelines on CLL

Inclusion Criteria:

Subjects with histologically confirmed chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), prolymphocytic leukemia (PLL)as defined by WHO classification of hematopoietic neoplasms, orRichter's transformation arising out of CLL/SLL and satisfying ≧1 of thefollowing conditions:

Progressive splenomegaly and/or lymphadenopathy identified by physicalexamination or radiographic studies

Anemia (<11 g/dL) or thrombocytopenia (<100,000/μL) due to bone marrowinvolvement

Presence of unintentional weight loss >10% over the preceding 6 months

NCI CTCAE Grade 2 or 3 fatigue

Fevers >100.5 degree or night sweats for >2 weeks without evidence ofinfection

Progressive lymphocytosis with an increase of >50% over a 2 month periodor an anticipated doubling time of <6 months

Need for cytoreduction prior to stem cell transplant

Subjects must have failed ≧2 prior therapies for CLL including anucleoside analog or ≧2 prior therapies not including nucleoside analogif there is a contraindication to such therapy

>10% expression of CD20 on tumor cells

ECOG performance status ≦2

Life expectancy ≧12 weeks

Subjects must have organ and marrow function as defined below:

Absolute neutrophil count (ANC) ≧1000/μL in the absence of bone marrowinvolvement Platelets ≧30,000/μL Total bilirubin ≦1.5×institutionalupper limit of normal unless due to Gilbert's disease AST(SGOT)≦2.5×institutional upper limit of normal unless due to infiltration ofthe liver Creatinine ≦2.0 mg/dL OR creatinine clearance ≧50 mL/min

No history of prior anaphylactic reaction to rituximab

No history of prior exposure to ofatumumab

Age ≧18 years

Body weight ≧40 kg

Able to swallow capsules without difficulty and no history ofmalabsorption syndrome, disease significantly affecting gastrointestinalfunction, or resection of the stomach or small bowel or ulcerativecolitis, symptomatic inflammatory bowel disease, or partial or completebowel obstruction

Exclusion Criteria:

A life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, interfere with the absorption or metabolism of Btkinhibitor PO, or put the study outcomes at undue risk

Any anticancer immunotherapy, chemotherapy, radiotherapy, orexperimental therapy within 4 weeks before first dose of study drug.Corticosteroids for disease-related symptoms are allowed provided 1 weekwashout occurs.

Active central nervous system (CNS) involvement by lymphoma

Major surgery within 4 weeks before first dose of study drug

Lactating or pregnant

History of prior malignancy, except for adequately treated basal cell orsquamous cell skin cancer, in situ cervical cancer, or other cancer fromwhich the subject has been disease free for at least 2 years or whichwill not limit survival to <2 years

History of Grade ≧2 toxicity (other than alopecia) continuing from prioranticancer therapy.

Results

6 Patients have been evaluated for DLT through end of cycle 2. 0 DLTsoccurred in these patients.

4 patients have had end of cycle 3 scans and blood counts. 3 of 4 areresponder per IWG criteria. Our response rate is 75% for these pts.

Example 15: Phase II Study of Btk Inhibitor+BR or FCR in R/R CLL

The purpose of this study is to establish the safety of orallyadministered Btk inhibitor in combination withfludarabine/cyclophosphamide/rituximab (FCR) and bendamustine/rituximab(BR) in patients with chronic lymphocytic leukemia (CLL)/smalllymphocytic lymphoma (SLL).

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Intervention: 420 mg/day of a Btk inhibitor, standard FCR or BR regimen

Applicable conditions: B-cell Chronic Lymphocytic Leukemia; SmallLymphocytic Lymphoma; Diffuse Well-differentiated Lymphocytic Lymphoma

Primary Outcome Measures:

To measure the number of participants with prolonged hematologictoxicity [Time Frame: 8 weeks from first dose]

Secondary Outcome Measures:

To measure the number of participants with adverse events as a measureof safety and tolerability [Time Frame: For 30 days after the last doseof Btk inhibitor]

To measure the number of patients who respond to treatment by measuringthe increase or decrease of disease in the lymph nodes and/or blood testresults [Time Frame: Patients may remain on study until the last subjectenrolled completes a maximum of 12 cycles of Btk inhibitor. Any subjectsstill receiving Btk inhibitor at that time may enroll in a long-termfollow-up study to continue to receive Btk inhibitor capsules]

Inclusion Criteria:

Histologically confirmed CLL or SLL and satisfying at least 1 of thefollowing criteria for requiring treatment:

Progressive splenomegaly and/or lymphadenopathy identified by physicalexamination or radiographic studies

Anemia (<11 g/dL) or thrombocytopenia (<100,000/μL) due to bone marrowinvolvement

Presence of unintentional weight loss >10% over the preceding 6 months

NCI CTCAE Grade 2 or 3 fatigue

Fevers >100.5° or night sweats for >2 weeks without evidence ofinfection

Progressive lymphocytosis with an increase of >50% over a 2 month periodor an anticipated doubling time of <6 months

1 to 3 prior treatment regimens for CLL/SLL

ECOG performance status of ≦1

≧18 years of age

Willing and able to participate in all required evaluations andprocedures in this study protocol including swallowing capsules withoutdifficulty

Ability to understand the purpose and risks of the study and providesigned and dated informed consent and authorization to use protectedhealth information (in accordance with national and local subjectprivacy regulations)

Exclusion Criteria:

Any chemotherapy, therapeutic antineoplastic antibodies (not includingradio- or toxin immunoconjugates), radiation therapy, or experimentalantineoplastic therapy within 4 weeks of first dose of study drug Radio-or toxin-conjugated antibody therapy within 10 weeks of first dose ofstudy drug

Concomitant use of medicines known to cause QT prolongation or torsadesde pointes

Transformed lymphoma or Richter's transformation

Any life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, interfere with the absorption or metabolism of Btkinhibitor PO, or put the study outcomes at undue risk

Any of the following laboratory abnormalities:

-   -   a. Absolute neutrophil count (ANC) <1000 cells/mm3 (1.0×109/L)    -   b. Platelet count <50,000/mm3 (50×109/L)    -   c. Serum aspartate transaminase (AST/SGOT) or alanine        transaminase (ALT/SGPT) ≧3.0×upper limit of normal (ULN)    -   d. Creatinine >2.0×ULN or creatinine clearance <40 mL/min

Example 16: Phase II Study of Btk Inhibitor in R/R DLBCL

The purpose of this study is to evaluate the efficacy of Btk inhibitorin relapsed/refractory de novo activated B-cell (ABC) and germinal-cellB-Cell (GCB) Diffuse Large B-cell Lymphoma (DLBCL).

Study Type: Interventional

Allocation: Non-Randomized

Endpoint Classification: Safety Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Intervention: 560 mg/day Btk inhibitor

Primary Outcome Measures:

To measure the number of patients with a response to study drug [TimeFrame: 24 weeks from first dose]

Participants will be followed until progression of disease or start ofanother anti-cancer treatment.

Secondary Outcome Measures:

To measure the number of patients with adverse events as a measure ofsafety and tolerability. [Time Frame: For 30 days after the last dose ofBtk inhibitor]

Participants will be followed until progression of the disease or startof another anticancer treatment.

To measure the number of participants pharmacokinetics to assist indetermining how the body responses to the study drug. [Time Frame:Procedure will be performed during the first month of receiving studydrug.]

Inclusion Criteria:

Men and women ≧18 years of age.

Eastern Cooperative Oncology Group (ECOG) performance status of ≦2.

Pathologically confirmed de novo DLBCL; subjects must have availablearchival tissue for central review to be eligible.

Relapsed or refractory disease, defined as either: 1) recurrence ofdisease after a complete remission (CR), or 2) partial response (PR),stable disease (SD), or progressive disease (PD) at completion of thetreatment regimen preceding entry to the study (residual disease):Subjects must have previously received an appropriate first-linetreatment regimen. Subjects with suspected residual disease after thetreatment regimen directly preceding study enrollment must have biopsydemonstration of residual DLBCL. Subjects who have not received highdose chemotherapy/autologous stem cell transplant (HDT/ASCT) must beineligible for HDT/ASCT as defined by meeting any of the followingcriteria: Age ≧70 years, Diffuse lung capacity for carbon monoxide(DLCO) <50% by pulmonary function test (PFT), Left ventricular ejectionfraction (LVEF) <50% by multiple gated acquisition(MUGA)/echocardiograph (ECHO), Other organ dysfunction or comorbiditiesprecluding the use of HDT/ASCT on the basis of unacceptable risk oftreatment-related morbidity, Subject refusal of HDT/ASCT.

Subjects must have ≧1 measurable (>2 cm in longest dimension) diseasesites on computed tomography (CT) scan.

Exclusion Criteria:

Transformed DLBCL or DLBCL with coexistent histologies (eg, follicularor mucosa-associated lymphoid tissue [MALT] lymphoma)

Primary mediastinal (thymic) large B-cell lymphoma (PMBL)

Known central nervous system (CNS) lymphoma

Any chemotherapy, external beam radiation therapy, or anticancerantibodies within 3 weeks of the first dose of study drug

Radio- or toxin-immunoconjugates within 10 weeks of the first dose ofstudy drug

Major surgery within 2 weeks of first dose of study drug

Any life-threatening illness, medical condition or organ systemdysfunction which, in the investigator's opinion, could compromise thesubject's safety, or put the study outcomes at undue risk

Clinically significant cardiovascular disease such as uncontrolled orsymptomatic arrhythmias, congestive heart failure, or myocardialinfarction within 6 months of screening, or any Class 3 or 4 cardiacdisease as defined by the New York Heart Association FunctionalClassification

Unable to swallow capsules or malabsorption syndrome, diseasesignificantly affecting gastrointestinal function, or resection of thestomach or small bowel or ulcerative colitis, symptomatic inflammatorybowel disease, or partial or complete bowel obstruction

Any of the following laboratory abnormalities:

-   -   a. Absolute neutrophil count (ANC) <750 cells/mm3 (0.75×109/L)        unless there is documented bone marrow involvement    -   b. Platelet count <50,000 cells/mm3 (50×109/L) independent of        transfusion support unless there is documented bone marrow        involvement    -   c. Serum aspartate transaminase (AST/SGOT) or alanine        transaminase (ALT/SGPT) ≧3.0 upper limit of normal (ULN)    -   d. Creatinine >2.0×ULN

Example 17: Assay of Drug Combinations

Combinations of a Btk inhibitor and additional cancer treatment agentswere assayed using DoHH2 cells.

DOHH2 is a DLBCL (diffuse large B-cell lymphoma) cell line, from atransformed follicular lymphoma patient. It is moderately sensitive to aBtk inhibitor.

The Btk inhibitor was incubated with other cancer drugs for 2 days.Assay was an alamar blue assay.

The combinations were:

a. Btk inhibitor and Gemicitabine;

b. Btk inhibitor and Dexamethasone;

c. Btk inhibitor and Lenalinomide;

d. Btk inhibitor and R-406;

e. Btk inhibitor and Temsirolimus;

f. Btk inhibitor and Carboplatin;

g. Btk inhibitor and Bortezomib; and

h. Btk inhibitor and Doxorubicin.

Results are presented in FIGS. 28-31.

Example 18: Assay of Drug Combinations

Combinations of a Btk inhibitor and additional cancer treatment agentswere assayed using TMD8 cells.

TMD8 is a NF-kB signalling-dependent ABC-DLBCL cell line. It issensitive to BTK inhibitors alone at low nanomolar concentrations (GI50˜1-3 nM). A Btk inhibitor was incubated with other cancer drugs for 2days. Assay was an alamar blue assay.

The combinations were:

a. Btk inhibitor and CAL-101;

b. Btk inhibitor and Lenalinomide;

c. Btk inhibitor and R-406;

d. Btk inhibitor and Bortezomib;

e. Btk inhibitor and Vincristine;

f. Btk inhibitor and Taxol;

g. Btk inhibitor and Fludarabine; and

h. Btk inhibitor and Doxorubicin.

Results are presented in FIGS. 32-39.

Example 19: Clinical Trial of Btk Inhibitor in Combination with BR

A clinical trial was performed to determine the effects of combining aBtk inhibitor with BR (bendamustine and rituximab). The Btk inhibitorwas administered. Following an increase in the concentration of lymphoidcells in the peripheral blood, BR was administered. Initial resultsindicated that the combination of the Btk inhibitor and BR resulted insubstantially no lymphoid cells in the peripheral blood.

Example 20: Clinical Trial of Btk Inhibitor in Combination withOfatumumab

A clinical trial was performed to determine the effects of combining aBtk inhibitor with ofatumumab. The Btk inhibitor was administered.Following an increase in the concentration of lymphoid cells in theperipheral blood, ofatumumab was administered. Initial results indicatedthat the combination of the Btk inhibitor and ofatumumab resulted in adecrease in lymphoid cells in the peripheral blood.

What is claimed is:
 1. A method for treating chronic lymphocyticleukemia in an individual who has failed at least one prior therapy forchronic lymphocytic leukemia comprising administering to the individualonce per day about 420 mg of an oral dose of an inhibitor of Bruton'styrosine kinase (Btk) having the structure:


2. The method of claim 1, wherein the oral dose is a capsule.
 3. Amethod for treating relapsed or refractory chronic lymphocytic leukemiain an individual comprising administering to the individual in needthereof once per day about 420 mg of an oral dose of an inhibitor ofBruton's tyrosine kinase (Btk) having the structure:


4. The method of claim 3, wherein the oral dose is a capsule.
 5. Themethod of claim 1, wherein, following administration of the inhibitor,the individual achieves a stable disease, a partial response, or acomplete response.
 6. The method of claim 1, wherein, followingadministration of the inhibitor, the individual achieves a partialresponse or a complete response.
 7. The method of claim 1, wherein,following administration of the inhibitor, the individual achieves acomplete response.
 8. The method of claim 1, wherein, followingadministration of the inhibitor, the individual does not experience aprogressive disease.
 9. The method of claim 1, wherein the individualhas nucleic acid deletion in chromosome
 17. 10. The method of claim 9,wherein the deletion is in 17p.
 11. The method of claim 10, wherein thedeletion is in 17p13.
 12. The method of claim 3, wherein, followingadministration of the inhibitor, the individual achieves a stabledisease, a partial response, or a complete response.
 13. The method ofclaim 3, wherein, following administration of the inhibitor, theindividual achieves a partial response or a complete response.
 14. Themethod of claim 3, wherein, following administration of the inhibitor,the individual achieves a complete response.
 15. The method of claim 3,wherein, following administration of the inhibitor, the individual doesnot experience a progressive disease.
 16. The method of claim 3, whereinthe individual has nucleic acid deletion in chromosome
 17. 17. Themethod of claim 16, wherein the deletion is in 17p.
 18. The method ofclaim 17, wherein the deletion is in 17p13.